28
73 CHAPTER 4 COLD - ADAPTATION OF HEAT - LABILE SHRIMP ALKALINE PHOSPHATASE Abstract Whatever their physicochemical environment, enzymes must always preserve an adequate catalytic efficiency, structural stability and regulatory sensitivity in order to function. Enzymes of organisms that live in cold habitats have generally been observed to have higher catalytic efficiency and lower heat stability. It has been proposed that cold-active enzymes need a degree of flexibility to prevent rigidity at low temperatures, while stabilizing interactions should prevent cold denaturation. In this study, sequences of alkaline phosphatases (APs) were studied and crystal structures of human placental AP (PLAP) and cold-active shrimp AP (SAP) were compared in order to identify features related to cold- adaptation for the AP family. Cold-active APs are found to be more negatively charged, have lower percentages of hydrophobic residues, higher percentages of polar residues, and lower arginine, glycine and proline content than their temperate homologues. Many structural features of SAP and PLAP have been preserved, such as the secondary structure content, the metal-binding triad, disulphide bridges, accessible surface area and the compactness of the structure, which are not likely to be involved in cold-adaptation. Conserved residues in mesophilic (temperate) APs were mainly located in the crown domain, the dimer interface and the wing regions, and are likely to be responsible for preserving structural elements and the subunit interface. The study suggests that heat-instability of cold-active AP structures mainly originates from a decrease of stabilizing hydrophobic residues in the protein core. Compared to SAP, PLAP has an extra bound metal and more prolines located in loops, which stabilize the structure. The two structures share common features with respect to disorder, although the flexible stretches in cold-active SAP were somewhat extended. Heat-instability might be slightly affected by the observed enhanced flexibility in the crown domain, since it is part of the dimer interface. The relative flexible regions can however not directly explain an improvement in catalytic efficiency, since they do not seem to influence the catalytic site. The catalytic efficiency is likely to be improved by the distribution of the excess negative charges: has optimized surface potentials to guide the substrate towards the active site. In addition, an alternative conformation of an active site arginine in SAP may facilitate the release of product and thereby increase the efficiency. Keywords: alkaline phosphatase, cold-adaptation, protein stability, catalytic efficiency.

CHAPTER 4 COLD ADAPTATION OF HEAT LABILE SHRIMP ALKALINE PHOSPHATASE · 2011-05-09 · Alkaline phosphatase amino acid sequences were retrieved from Swissprot and TrEMBL (release

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Page 1: CHAPTER 4 COLD ADAPTATION OF HEAT LABILE SHRIMP ALKALINE PHOSPHATASE · 2011-05-09 · Alkaline phosphatase amino acid sequences were retrieved from Swissprot and TrEMBL (release

7 3

C H A P T E R 4

C O L D - A D A P T A T I O N

O F H E A T - L A B I L E S H R I M P A L K A L I N E P H O S P H A T A S E Abstract Whatever their physicochemical environment, enzymes must always preserve an adequate catalytic efficiency, structural stability and regulatory sensitivity in order to function. Enzymes of organisms that live in cold habitats have generally been observed to have higher catalytic efficiency and lower heat stability. It has been proposed that cold-active enzymes need a degree of flexibility to prevent rigidity at low temperatures, while stabilizing interactions should prevent cold denaturation. In this study, sequences of alkaline phosphatases (APs) were studied and crystal structures of human placental AP (PLAP) and cold-active shrimp AP (SAP) were compared in order to identify features related to cold-adaptation for the AP family. Cold-active APs are found to be more negatively charged, have lower percentages of hydrophobic residues, higher percentages of polar residues, and lower arginine, glycine and proline content than their temperate homologues. Many structural features of SAP and PLAP have been preserved, such as the secondary structure content, the metal-binding triad, disulphide bridges, accessible surface area and the compactness of the structure, which are not likely to be involved in cold-adaptation. Conserved residues in mesophilic (temperate) APs were mainly located in the crown domain, the dimer interface and the wing regions, and are likely to be responsible for preserving structural elements and the subunit interface. The study suggests that heat-instability of cold-active AP structures mainly originates from a decrease of stabilizing hydrophobic residues in the protein core. Compared to SAP, PLAP has an extra bound metal and more prolines located in loops, which stabilize the structure. The two structures share common features with respect to disorder, although the flexible stretches in cold-active SAP were somewhat extended. Heat-instability might be slightly affected by the observed enhanced flexibility in the crown domain, since it is part of the dimer interface. The relative flexible regions can however not directly explain an improvement in catalytic efficiency, since they do not seem to influence the catalytic site. The catalytic efficiency is likely to be improved by the distribution of the excess negative charges: has optimized surface potentials to guide the substrate towards the active site. In addition, an alternative conformation of an active site arginine in SAP may facilitate the release of product and thereby increase the efficiency. Keywords: alkaline phosphatase, cold-adaptation, protein stability, catalytic efficiency.

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4.1 INTRODUCTION 4 . 1 . 1 E n z y m e s a t L o w T e m p e r a t u r e s Organisms have evolved to live in environments with low temperatures. In general terms, species that live in cold environments are called cold-adapted, cold-active or psychrotolerant species. When their optimum growing temperature is below 20ºC, they are referred to as psychrophiles. Organisms that live in cold environments must contain proteins that have evolved to compensate for the reduction in reaction rates. Several cold-adapted enzymes have, in fact, been shown to reduce the activation free energy for the reaction catalyzed (Davail 1994; Iyo 1999; Low 1973). Enzymes from psychrophilic organisms are characterized by an increased catalytic efficiency at lower temperature, usually accompanied by a reduced thermal stability and a lower temperature optimum, than their mesophilic counterparts. The catalytic efficiency is expressed as the ratio of the turnover number kcat and the Michaelis Menten constant KM: kcat/KM (s-1M-1). Cold-active enzymes can improve the catalytic efficiency by increasing the turnover number or by improving substrate-binding interactions to lower the value of KM. Although not proven, a generally accepted hypothesis states that the observed reduced thermal stability arises from increased local molecular flexibility, which would prevent enzymes from becoming overly rigid at low temperatures, and thus unable to function (Somero 1975). For general reviews on the cold-adaptation of proteins, see e.g. Feller 1996, Feller 1997, Gerday 1997, Marshall 1997 and Smalås 2000. Proteins from psychrophilic species have been shown to be more susceptible to heat denaturation than their mesophilic homologues. This observation must be reflected in their structures. The strengths of electrostatic interactions, hydrogen bonds and Van der Waals interactions increase as temperature decreases, while the hydrophobic entropy effect decrease at lower temperatures. The influence of temperature on protein structure and stability is described in section 1.2.2. A brief summary with possible characteristics of cold-active structures is listed below:

• Less arginines and a lower ratio Arg/(Arg+Lys) • Less tight packing of hydrophobic residues in the interior of the structure, which may be reflected in a lower (I+L) / (I+L+V) ratio. • Larger surface to volume ratio, more internal cavities. • Less charged and polar residues located at the water-accessible surface. • Larger degree of flexibility, which accomplished by the presence of glycines in loops, lack of local ion-pair networks or disulfide bridges, or a reduction of interdomain interactions.

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4 . 1 . 2 C o l d - Ac t i v e Al k a l i n e P h o s p h a t a s e s Alkaline phosphatases (abbreviated as AP; E.C. 3.1.3.1.) are abundant in a wide range of prokaryotic and eukaryotic species. They are usually homodimeric and catalyze the hydrolysis or, in the presence of a phosphate acceptor, the transphosphorylation, of a wide variety of phosphate monoesters to yield inorganic phosphate and an alcohol or a new monoester. The one product, inorganic phosphate, is also an inhibitor for the enzyme (Fernley 1967). The catalytic reaction mechanism has been elucidated for E.coli AP. The rate-determining step for hydrolysis at alkaline pH is the release of inorganic phosphate (Hull 1976). Cold-active APs of the following species have been studied: gadus morhua (Atlantic cod) (Ásgeirsson 1995), marine Vibrio sp (Hauksson 2000), a psychrophilic bacterial strain TAB5 (Rina 2000), an Antarctic bacterial strain HK47 (Kobori 1984), and Pandalus borealis (Arctic shrimp) (Nilsen 2001; Olsen 1991). Heat-labile APs from Sphingobacterium antarcticus, Arthrobacter D10 and Shewanella sp have also been partially purified and characterized. The enzymes from vibrio sp and HK47 are extremely heat-labile with half-lives of 6 and 2 minutes at 40ºC respectively (Hauksson 2000; Kobori 1984); the cod and shrimp enzymes are somewhat more heat-resistant, with inactivation after 1.2 and 15 minutes at 65ºC, respectively (Ásgeirsson 1995; Olsen 1991). The known temperature optima for catalysis of the cold-active APs are well above the temperatures of their natural environments, 25ºC for TAB5 and HK47; 37ºC for shrimp. This implies that cold-active APs could function at similar temperatures to their mesophilic counterparts, but retain a higher residual activity at lower temperatures.

The specific activities of cod, shrimp and vibrio sp. AP are in the same order of magnitude as mammalian calf AP; between 2000 and 4500 U mg-1 at 25ºC (cod and vibrio) or 37ºC (shrimp and calf), depending on buffer composition (Ásgeirsson 1995; Biotec 2000; Hauksson 2000; Olsen 1991). Cold-active cod AP has a higher catalytic efficiency than the mesophilic calf AP at 5°C (kcat/KM of 31·105 and 12·105 s-1M-1 respectively), which arises from an improved turnover number. The activation energy was found to be lower for the cod enzyme. Moreover, cod AP hydrolyzes several substrates at different reaction rates than its bovine homologue, indicating differences in both substrate-specificity and in details of the kinetic mechanism of catalysis (Ásgeirsson 1995). For example, cold-active cod and marine vibrio AP have considerably lower affinities for phosphate than the calf enzyme (Ásgeirsson 1995; Hauksson 2000), resulting in a more facile release of product. Since product release is the rate-limiting step in the reaction cycle at alkaline pH (Hull 1976), this explains the improved turnover for these cold-active APs. In addition, the stability of the monomer and the dimeric structure were found to be less stable than its mammalian counterpart.

The structure of shrimp AP (SAP) has been solved (chapter 2) and is compared with the structure of mesophilic human placental AP (PLAP) in section 4.3.2. SAP operates at about 5ºC in vivo, has a high catalytic efficiency and is heat labile. SAP is not strictly psychrophilic with a kinetic optimum temperature for catalysis being the same as for mesophilic homologues, 37ºC (Lustig 1971; Olsen 1991), but can be considered a cold-active enzyme. On the other hand, the optimal temperature of enzymatic activity is usually not the same as the optimal growth temperature of the organism, which is dependent on many parameters (Gerday 1997; Sheridan 2000).

APs are usually functional dimers. ECAP has been reported to exhibit negative cooperativity in substrate hydrolysis and intragenic complementation (i.e. the combination of differently mutated monomeric APs results in a heterodimer with a higher level of activity than would be expected on the activities of the individual monomers), and PLAP has been reported to display allostery (Hoylaerts 1997). This implies communication between the monomers, most likely via the dimer interface. Interface interactions enhance

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stability of the tertiary and quaternary structure (Martin 1999). Dissociation of the dimer induces conformational changes in the monomers, which affect the metal-binding sites and hence catalytic activity (Falk 1982). Most APs loose activity when the dimer dissociates (Ásgeirsson 2000; Bortolato 1999; Falk 1982), but the dissociated subunits of SAP have been reported to retain enzyme activity (Olsen 1991). This may imply that the intersubunit interactions for SAP are less crucial for activity than for mammalian or bacterial APs. In this chapter, sequences and structural features that may explain the characteristics of cold-active alkaline phosphatases are studied. Comparisons are based on sequence alignments of thermophilic, mesophilic and cold-active AP sequences (described in sections 4.2.1 and 4.3.1). In addition, the crystal structures of mesophilic PLAP and cold-active SAP are compared on different structural properties (sections 4.2.2 and 4.3.2). In the discussion (section 4.4) the observed analogies and differences are discussed.

4.2 METHODS 4 . 2 . 1 P r i m a r y S e q u e n c e An a l y s i s Alkaline phosphatase amino acid sequences were retrieved from Swissprot and TrEMBL (release 40.12 of March 5 2002; release 19.10 of March 1, 2002, respectively), and limited to one sequence per species. Initially, six thermophilic, thirty-nine mesophilic and three cold-active AP sequences were found. In order to find characteristics that could be indicative of cold-adaptation, sequences with less than twenty percent amino acid identity to the SAP (“Pandalus borealis”) sequence were rejected. Thus one thermophilic sequence was rejected, while all cold-active sequences were kept. The mesophilic sequences, of which there are many, were subjected to a higher rejection criterion, retaining only sequences with more than thirty percent identity to SAP. This resulted in five thermophilic, twelve mesophilic and three cold-active AP sequences. The isoelectric points were calculated with PROTPARAM (available at http://www.expasy.ch). Each sequence was aligned with the shrimp AP sequence using CLUSTALW (Thompson 1994) at PBIL (http://npsa-pbil.ibcp.fr), which computed the percentages of identical and strongly similar amino acids among the sequences. The resulting sequence identity was used as an additional criterion to accept or reject the sequence. Net charge was calculated by subtracting the number of aspartates and glutamates from the number of arginines and lysines. Amino acids LIVGAFCM were considered hydrophobic; CHNQSTY were defined as polar; FYW as aromatic and RKDE were considered charged. Conservation of Residue Types Within Three Temperature Categories All selected AP sequences, assigned as either thermophilic, mesophilic or psychrophilic, were aligned with ClustalW. The alignments were manually adjusted and inspected for conservation of residue types within each temperature category. When inspecting the aligned sequences, the following philosophy was used: residue types that are conserved among all temperature categories, or among mesophilic and psychrophilic sequences, were regarded as not involved in cold-adaptation. The limited number of available psychrophilic and thermophilic sequences forced the following strategy to be used to identify residues that may be involved in cold-adaptation: residue types that are conserved in more than half of the mesophilic sequences, and not in

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4 . 3 R E S U L T S - 7 7

psychrophilic or thermophilic sequences, were marked as “conserved mesophilic residues”. These residues are thus only conserved within mesophilic AP species and are likely to be involved in adaptation to varying temperatures. 4 . 2 . 2 S t r u c t u r a l C o m p a r i s o n s The coordinates of shrimp and human AP are available at the protein Data Bank with entry codes 1K7H and 1EW2, respectively. The definitions of secondary structural elements used are described in the header of the PDB coordinate files. The structures were superimposed using LSQMAN (Kleywegt 1994b). Hydrogen bonds were calculated with HBPLUS Hydrogen Bond Calculator version 3.15 (McDonald 1994) with the default criteria. Ion pairs were defined as non-directional interactions of two oppositely charged atoms from Asp/Glu and Arg/Lys/His with interatomic distances less than 4.0Å. They were calculated with CONTACT (Skarzynski 1988) of the CCP4 suite. Manual inspection of this selection found salt bridges, which were defined as direct interactions of a pair of Asp/Glu side chain carboxyl oxygen atom and a Arg/Lys/His side chain nitrogen atom with interactomic distances less than 3.0Å (Kumar 2001). The residues participating in the dimer interface were calculated with DIMPLOT (Wallace 1995).

The accessible surface area was calculated and displayed using GRASP (Nicholls 1991). The protein’s volume was calculated as the volume inside the molecular surface. The buried surface area was calculated as twice the molecular surface of the monomer minus the molecular surface of the dimer, divided by 2. Residues with a solvent accessible area smaller than 10 Å2 were considered internal. The solvent accessible areas per atom were calculated by AREAIMOL (CCP4) and voids were calculated with VOIDOO (Kleywegt 1994a).

4.3 RESULTS 4 . 3 . 1 C o m p a r i s o n o f P r i m a r y S e q u e n c e s Sequences of alkaline phosphatases that fulfilled the requirements (described in section 4.2.1) were grouped in the three classes thermophilic, mesophilic and cold-active, according to the growth temperature of the organisms. This yielded five thermophilic, twelve mesophilic and three cold-active AP sequences. This relatively small number of sequences is marginal for statistical sequence analysis. Nevertheless, the sequences may show trends that explain adaptive strategies of the alkaline phosphatase family to the cold. For convenience, the sequences of cold-adapted organisms will be referred to as psychrophilic, although SAP is cold-adapted rather than strictly psychrophilic. The sequence numbering of SAP is conform to the Protein Data Bank entry 1K7H. In table 4.1 the amino acid composition of the sequences are shown; in table 4.2 calculated properties of the sequences are listed. The following trends are observed:

• The calculated pI of the protein sequences decrease from thermophilic to mesophilic to psychrophilic enzymes. • The percentage of hydrophobic residues in psychrophilic APs is lower than in the mesophilic or thermophilic sequences. For the alkaline phosphatase family, the mean percentage of leucine and

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7 8 - C H A P T E R 4

valine residues decrease from thermophiles to psychrophiles, while the mean percentage of isoleucines is highest for psychrophiles and lowest for thermophiles. As a result, the (I+L)/(V+I+L) ratio is similar for thermophiles and mesophiles and highest for psychrophiles, indicating that APs do not use the strategy of decreasing the length of aliphatic side chains in order to influence structure stability. • The percentage of polar residues increases slightly from thermophilic to psychrophilic enzymes, while the percentage of charged residues fluctuates: thermophilic sequences posses the highest mean percentage of positively charged residues, psychrophilic sequences have the highest percentage of negatively charged residues. A structure with more polar or charged side chains could in principle form more ion pairs or hydrogen bonds. Therefore it is not straightforward to relate the percentages of polar residues with protein stability and it is important to know whether these amino acids are located in the interior or on the surface of the protein. • The percentage of aromatic residues does not display an increasing or decreasing trend with the growth temperature of the organisms: the percentage is lowest for mesophiles and highest for psychrophiles. • The mean percentage of arginines decreases from thermophilic to psychrophilic sequences. This trend is expected, since arginine is a stabilizing amino acid. The R/(R+K) ratio, a possible indicator for cold-adaptation at the level of the primary structure, decreases as well: from 0.58 (thermophiles) to 0.31 (psychrophiles). This trend has been observed for other protein families. • The psychrophilic enzymes have the lowest percentage of the conformationally restricted prolines, but also the lowest percentage of glycines. • The mean percentage of cysteines, that potentially can form disulfide bridges, is expected to decrease from thermophilic to psychrophilic proteins. The AP family does not follow this trend; the mesophilic sequences possess the most cysteines. Other observed trends are the increase of serine and threonine residues.

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4 . 3 R E S U L T S - 7 9

Table 4.1a Amino acid composition of alkaline phosphatase sequences from thermophilic species, in percentages. organism G

A V I L P

M

C

F Y W H R K D E N Q S T

P.abyssi 9.3 7.1 7.9 8.5 10.3 2.0 3.4 0.2 3.6 2.8 0.0 2.8 4.2 5.9 7.3 7.7 3.6 1.0 5.7 6.7 T.maritima 7.6 6.9 7.8 6.5 9.9 4.1 2.8 0.2 4.4 3.2 1.4 1.6 3.7 7.6 6.5 6.9 3.9 1.6 6.2 7.1 T.aquaticus 9.4 12.8 8.2 2.6 10.8 4.4 1.8 0.2 3.6 3.4 1.2 2.4 8.2 3.2 4.8 6.4 3.6 4.0 4.2 4.2 T.sp. 9.8 13.0 8.2 2.8 11.0 4.4 1.8 0.2 3.4 3.8 1.2 2.2 7.8 3.2 4.8 5.8 3.8 4.2 4.6 4.6 T.thermo-philus

5.7 12.8 8.2 2.4 11.2 3.4 3.4 0.0 3.6 3.6 1.0 2.4 8.4 3.0 4.6 6.4 3.8 4.0 4.2 4.4

Spread 5.7 9.8

6.9 13.0

7.8 8.2

2.4 8.5

9.9 11.2

2.0 4.4

1.8 3.4

0.0 0.2

3.4 4.4

2.8 3.8

0.0 1.4

1.6 2.8

3.7 8.4

3.0 7.6

4.6 7.3

5.8 7.7

3.6 3.9

1.0 4.2

4.2 6.2

4.2 7.1

Mean 8.4 10.5 8.1 4.6 10.6 3.7 2.6 0.2 3.7 3.4 1.0 2.3 6.5 4.6 5.6 6.6 3.7 3.0 5.0 5.5

Table 4.1b Amino acid composition of alkaline phosphatase sequences from mesophilic species, in percentages. organism G

A V I L P

M C F Y W H R K D E N Q S T

B.mori 10.2 11.0 7.9 2.4 8.0 4.2 2.0 1.6 3.5 1.8 1.6 4.0 6.8 2.9 5.7 6.6 3.5 3.5 5.3 7.5B.taurus 8.6 9.4 6.7 3.8 9.2 5.0 3.2 1.1 2.9 3.8 1.0 4.0 3.6 5.5 5.5 5.2 5.2 3.6 6.5 6.3C.familiaris 9.0 9.2 7.4 3.8 8.6 4.8 3.2 1.0 2.6 4.2 1.0 4.6 4.0 5.6 5.8 5.2 5.4 3.2 5.0 6.8C.intestinalis 8.3 10.4 6.2 5.7 9.3 2.8 2.9 1.4 3.1 3.1 0.7 2.9 3.1 5.9 6.2 5.4 5.0 4.5 6.6 6.6D.melano-gaster

8.8 8.8 6.6 5.5 6.9 5.5 2.8 0.5 3.3 3.5 1.7 4.0 5.5 4.2 5.4 6.6 5.2 3.1 4.7 7.4

F.catus 9.0 8.6 7.3 4.0 8.8 5.0 3.2 1.1 2.9 3.8 1.0 4.2 4.0 5.3 5.3 5.3 5.2 3.2 5.9 6.9G.gallus 8.7 11.0 7.7 2.9 10.8 4.8 2.1 1.3 2.7 3.1 1.0 3.7 6.4 4.4 5.8 5.8 3.9 3.9 4.2 6.0H.roretzi 7.9 7.8 6.0 6.0 7.5 3.1 3.1 1.5 3.3 4.1 0.7 3.0 4.0 6.3 6.1 6.3 6.8 2.8 7.0 6.8H.sapiens 9.2 11.0 6.2 3.4 10.8 5.8 2.8 1.1 3.2 3.2 0.7 2.8 5.8 3.7 5.8 5.6 3.0 3.7 5.4 6.7M.musculus 8.7 10.8 6.6 3.6 8.3 4.7 4.0 1.7 3.2 2.8 1.1 3.2 4.0 4.7 4.6 4.9 3.6 5.1 7.2 6.8R.solana-cearum

12.6 14.3 8.3 2.5 8.1 3.3 2.7 0.6 3.3 2.9 0.0 2.5 3.9 7.3 7.0 3.5 3.3 1.2 5.0 10.6

R.norvegicus 8.3 9.3 6.9 3.4 9.1 6.2 2.4 0.9 3.1 3.8 1.3 2.7 3.8 4.0 5.3 4.4 4.0 4.9 6.0 10.3

Spread 7.9 12.6

7.8 14.3

6.0 8.3

2.4 6.0

6.9 10.8

3.1 6.2

2.04.0

0.51.7

2.63.5

1.84.2

0.01.7

2.54.6

3.66.8

2.97.3

4.6 7.0

3.5 6.6

3.0 6.8

1.25.1

4.27.2

6.010.

6Mean 9.1 10.3 7.0 3.9 8.8 4.6 2.9 1.2 3.1 3.3 1.0 3.5 4.6 5.0 5.7 5.4 4.5 3.6 5.7 7.4

Table 4.1c Amino acid composition of alkaline phosphatase sequences from psychrophilic species, in percentages. organism G A V I L P M C F Y W H R K D E N Q S T

P.borealis 8.1 8.9 5.5 5.5 7.6 2.8 2.1 0.6 4.0 4.0 1.5 3.4 4.9 4.5 10.0 7.0 3.4 2.3 4.7 9.1TAB5 8.5 9.6 4.0 8.0 7.7 2.1 1.9 0.5 6.4 2.7 0.5 1.6 1.6 7.7 5.9 5.3 5.9 1.9 9.1 9.1V.sp 7.7 9.0 5.2 5.2 8.6 3.8 2.5 0.2 3.8 3.6 1.0 2.7 2.5 7.5 6.5 6.3 5.4 5.0 7.3 6.1Spread 7.7

8.5 8.9 9.6

4.0 5.5

5.2 8.0

7.6 8.6

2.1 3.8

1.92.5

0.20.6

3.86.4

2.73.6

0.51.5

1.63.4

1.64.9

4.57.7

5.9 10.0

5.3 7.0

3.4 5.9

1.95.0

4.79.1

6.19.1

Mean 8.1 9.2 4.9 6.2 8.0 2.9 2.2 0.4 4.7 3.4 1.0 2.6 3.0 6.6 7.5 6.2 4.9 3.1 7.0 8.1

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Table 4.2a Analysis of alkaline phosphatase sequences from thermophilic species. Organism SwissProt/

TrEMBL ID MW (kD)

pI net charge

hydro- phobic

%

polar

%

pos.

%

neg.

%

aro-matic

%

R / (R+K)

(L+I) / (L+I+V)

identity to SAP

%

similarityto SAP

% Pyrococcus.abyssi Q9UZV2 54.2 5.0 -24 46.7 57.7 9.8 15.0 6.4 0.42 0.70 23.9 13.8Thermotoga.maritima Q9WYO3 48.2 5.5 -9 41.7 56.2 11.3 13.4 9.0 0.33 0.68 27.6 18.1Thermus.aquaticus Q9RA56 54.7 7.9 +1 45.8 54.6 11.4 11.2 8.2 0.72 0.62 20.3 16.6Thermus.sp. 086025 54.7 8.4 +2 46.8 54.2 11.0 10.6 8.4 0.71 0.63 19.7 16.1Thermus.thermo-philus

Q934S9 54.8 8.4 +2 43.7 54.0 11.4 11.0 8.2 0.74 0.62 20.3 16.6

Spread 48.2 54.8

5.0 8.4

2-24

41.746.8

54.657.7

9.811.4

10.615.0

6.49.0

0.33 0.74

0.62 0.70

19.727.6

13.818.1

Mean 53.3 7.0 -5.6 44.9 55.3 11.0 12.2 8.0 0.58 0.65 22.4 16.2

Table 4.2b Analysis of alkaline phosphatase sequences from mesophilic species. organism SwissProt/

TrEMBL ID MW (kD)

pI net charge

hydro-phobic

%

polar

%

pos.

%

neg.

%

aro-matic

%

R / (R+K)

(L+I) / (L+I+V)

identity to SAP

%

similarityto SAP

% Bombyx.mori P29523 59.2 5.9 -14 43.1 59.4 9.7 12.3 6.9 0.70 0.57 37.4 19.0Bos.taurus P09487 57.2 6.2 - 8 42.0 58.8 9.1 10.7 7.7 0.40 0.66 40.5 19.0Canis.familiaris Q9N0V0 55.1 6.4 +7 42.2 59.4 9.6 11.0 7.8 0.42 0.63 42.0 19.7Ciona.intestinalis Q9NL48 62.8 5.6 -15 44.2 58.9 9.0 11.6 6.9 0.34 0.71 33.4 20.2Drosophila.melano-gaster

Q24238 64.0 6.0 -13 39.9 58.9 9.7 12.0 8.5 0.57 0.65 33.6 17.4

Felis.silvestris.catus Q29486 57.3 6.3 - 7 42.0 59.2 9.3 10.6 7.7 0.43 0.64 39.7 20.0Gallus.gallus Q92058 56.8 6.6 - 4 44.5 57.0 10.8 11.6 6.8 0.59 0.64 40.7 18.8Halocynthia.roretzi Q94581 66.8 5.8 -13 39.8 62.5 10.3 12.4 8.1 0.39 0.69 34.0 15.6Homo sapiens PPB1 58.0 5.9 +10 44.5 56.1 9.5 11.4 7.1 0.61 0.70 36.3 17.6Mus.musculus P24823 57.2 6.3 - 6 43.7 57.7 8.7 9.5 7.1 0.46 0.64 36.1 18.9Ralstonia.solana-cearum

Q8Y383 49.6 5.6 -11 49.1 57.5 11.2 10.5 6.2 0.35 0.56 30.1 17.9

Rattus norvegicus P51740 59.8 5.8 +10 40.3 58.3 7.8 9.7 8.2 0.49 0.64 33.6 18.1Spread 49.6

66.8 5.6 6.6

+10-14

39.849.1

56.162.5

8.711.2

9.712.4

6.28.5

0.34 0.70

0.56 0.71

30.142.0

15.620.2

Mean 58.7 6.0 -5.3 50.0 58.6 9.6 11.1 7.4 0.48 0.64 36.5 18.5

Table 4.2c Analysis of alkaline phosphatase sequences from psychrophilic species. organism SwissProt/

TrEMBL ID MW kD

pI net charge

hydro-phobic

%

polar

%

pos.

%

neg.

%

aro-matic

%

R / (R+K)

(L+I) / (L+I+V)

identity to SAP

%

similarityto SAP

% Pandalus.borealis Q9BHT8 52.5 4.7 -36 38.3 62.1 9.4 17.0 9.5 0.52 0.70 100 -TAB5 Q9KWY4 40.4 5.5 -7 40.2 59.8 9.3 11.2 9.6 0.17 0.80 21.6 19.5Vibrio sp Q93P54 57.4 5.5 -15 38.4 60.9 10.0 12.8 8.4 0.25 0.73 22.3 16.7Spread 40.4

57.4 4.7 5.5

-7-36

38.340.2

59.862.1

9.310.0

11.217.0

8.49.6

0.25 0.52

0.70 0.80

21.622.3

16.719.5

Mean 50.1 5.2 -19.3 39.0 60.9 9.6 13.7 9.2 0.31 0.74 22.0 18.1

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4 . 3 R E S U L T S - 8 1

Conservation of Residue Types Within Three Temperature Categories The aligned and classified AP sequences were inspected for conservation of residue types within a temperature category and between temperature categories. Within a given temperature-category, the sequences show conservations. Some residues types are even present among different temperature categories, but few amino acids are absolutely conserved in all sequences: S86 (the catalytically active serine), D315 and H319 (ligands for Zn1 in the active site), D356 and H357 (ligands for Zn2), H149 and E310 (ligands for Zn3), T76, G96, G140, A150, P152, G202, G203, L287, A294, L298, I314, G443. Note that although these residues are conserved among the sequences selected in this study, they are not necessarily conserved in all AP sequences presently known. Conserved mesophilic residues (see Methods section) were highlighted in the structure of human placental AP and appeared to be localized as follows (see figure 4.1):

• Two loops of the crown domain are quite conserved, including residues that are part of the dimer interface; • Several clusters in the wing regions are conserved, probably to maintain the integrity of the local structure; • A loop connecting the helix that runs parallel to the central β-sheet with a strand. This loop includes a few residues that participate in the dimer interface and a residue that interacts with the crown domain; • A stretch of twelve residues near the interface, of which five residues participate in dimer formation. The regions that hardly contain conserved mesophilic residues are the N-terminal helix, one loop of the crown domain, the central β-sheet and the immediate vicinity of active site (except Gly358).

Figure 4.1 Conserved mesophilic residues are represented as spheres in the structure of human placental AP. The figure has been

prepared with MOLSCRIPT (Esnouf 1997) and RASTER3D (Merritt 1997).

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8 2 - C H A P T E R 4

4 . 3 . 2 S t r u c t u r a l C o m p a r i s o n o f a M e s o p h i l i c a n d a C o l d - Ac t i v e AP In order to identify structural adjustments to cold-adaptation in the alkaline phosphatase family, two crystal structures are compared. The mesophilic structure of human placental AP, PLAP, has been solved to a resolution of 1.82Å with 479 residues per monomer (Le Du 2001); the cold-active structure of shrimp AP, SAP, has been determined to a resolution of 1.92Å with 476 residues per monomer (De Backer 2002). The overall structures are very similar; they superimpose with a root mean square deviation of 1.07Å for 455 Cα-atoms (figure 4.2). A third available crystal structure of E.coli AP is not considered because of its low sequence homology with SAP. Primary Structure The sequences of SAP (sequence identity Q9BHT8) and PLAP (PPB1) are identical for 36.3% of their residues. In addition, 17.6% of the amino acids types are strongly similar, according to the classifications of ClustalW. A sequence alignment is shown in appendix A. The “Pandalus borealis” entry in tables 1c and 2c will be referred to as “SAP”; the “Homo sapiens” entry in tables 1b and 2b are from the human placenta (PLAP). The two sequences follow the general trends as observed for the comparison between mesophilic and psychrophilic AP sequences as described section 4.3.1.

In the AP family, mesophilic enzymes have the highest percentages of glycine and proline residues. Indeed, PLAP has 43 glycines and 26 prolines, compared to 38 glycines and 13 prolines for SAP. Of these residues, 28 glycines and 5 prolines occupy similar positions in the two structures and cannot be involved in cold-adaptation. Of the remaining “unique” residues, SAP has 4 glycines and 3 prolines located in helices, 2 glycines and no prolines in strands, 4 glycines and 6 prolines in coil regions; PLAP has 6 glycines and 3 prolines in helices; no glycines or prolines in strands and 9 glycines and 18 prolines in coils (table 4.3). In summary, PLAP has more unique glycines in helices and coil regions, while SAP has more glycines in strands. Both enzymes have the same number of prolines located in helices, and none in strands, while PLAP has twelve more prolines located in coil regions. The stabilizing effect of these prolines must rigidify the PLAP structure to some extent.

Table 4.3 Distribution of glycine and proline residues in secondary structural elements of PLAP and SAP.

Helices Strands Coils

glycines prolines glycines prolines glycines prolines

SAP 4 3 2 0 4 6

PLAP 6 3 0 0 9 18

The 28 glycine and 5 proline residues that occupy similar positions in the structures are not taken into account in this table.

Residue types on the surface and in the interior of the structures were also investigated. Residues with a solvent accessible area smaller than 10 Å2 were defined as internal, others external. The residues were further divided into polar, hydrophobic and aromatic groups (table 4.4). SAP has more polar and aromatic residues located at the interior and exterior of the structure than PLAP. Of the polar residues, SAP has far more negatively charged residues at the surface than PLAP. By contrast, PLAP has more hydrophobic residues at the interior and the exterior of the structure.

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Of the 50 "conserved mesophilic residues" (see Methods section), 26 were polar, 20 hydrophobic and 4 were prolines. They were compared to the corresponding residues in the sequence of SAP (table 4.5) and were substituted for 32 polar amino acids, 16 hydrophobic residues, 1 proline and 1 tryptophan.

Table 4.4 Internal and external residues divided into residue categories.

SAP PLAP

internal external internal external

hydrophobic 134 68 139 78

aromatic 21 24 17 22

polar 106 190 100 180

of which positively charged 5 38 6 43

of which negatively charged 16 68 15 44

Residues with a solvent accessible area smaller than 10 Å2 are considered internal, residues with higher accessible areas are

considered external. Amino acids LIVGAFCM were considered hydrophobic, FYW aromatic, CDEGHRKNQSTY polar; RK as

positively charged and DE as negatively charged.

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Res.

Nr.

meso-

philic

a.a.

psychro-

philic

a.a.

thermo-

philic

a.a.

Structural

element

dimer

inter-

face

int/

ext

Res.

Nr.

meso-

philic

a.a.

psychro-

philic

a.a.

thermo-

philic

a.a.

Structural

element

dimer

inter-

face

int/

ext

53 Q GGK RRH helix - E 224 L ERR KD- - - E

59 G E-- L-- helix Y I 234 K LLA KAA helix - E

60 E RTT A-- helix Y E 261 V TSL --- - - E

62 T,S KNI LLL* - near E 269 E SLG AAA* - - E

63 L* IYY FRM helix near I 296 R EQN PEE helix - E

65 M WQL LKD helix near I 303 KR NSD GNE - - E

84 P TTV TTT* - near I 320 D,E ASA DAL helix - E

107 S,T D-- --- - Y I 321 G NNN* NNN* - - I

117 T,S YAH PLV helix - E 322 K,R QND DDD* helix - E

118 T,S QVV VVL helix - I 326 A* SLM TIV helix - I

122 G S-- --- helix Y E 330 A TIL VVT helix - I

123 N L-- --- helix - E 358 S,T GEE EEA sheet - I

124 E F-- - helix Y E 360 V TGG GGG* - Y- I

125 V T-- L - - I 361 F LGS GGV sheet Y I

159 S VAQ VVN - - I 362 T,S TFF LLG sheet Y I

165 Y EEE* --- - - E 363 F ITG GGG* sheet Y I

166 S NEN --- helix - I 364 G TLF LLI - Y I

170 M V-- --- helix - I 370 G ND- S-- - - E

171 P V-- --- helix - E 371 N TER VVV* - Y E

174 A R-- --- - - E 374 F LSE DDL - - I

179 G I-- EEE* - - E 375 G* DEA ESK - - E

190 N RST NHF helix - E 378 P GDD RRN sheet - E

195 D N-- - helix - E 386 P RE- KDQ sheet - E

217 Y DE- --- - - E 389 S,T IA- KDE - Y I

220 D,E PK- --- - - E 394 N SK- MHY - - I

In column 2 the conserved mesophilic residue type is indicated, corresponding to the residue mentioned in column 1. Columns 3

and 4 list the corresponding amino acids in psychrophilic (the residue type of SAP is underlined) and thermophilic amino acid

sequences. An asterisk indicates amino acid identity for all entries. For each residue the secondary structural element (helix,

strand or neither) is indicated, if it participates in the dimer interface and whether it is located at the interior (I) or exterior (E) of the

structure.

Table 4.5 Conserved mesophilic residues and the corresponding amino acids in psychrophilic and mesophilic APs.

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Secondary Structure The structures of cold-active shrimp alkaline phosphatase and human placental alkaline phosphatase superimpose well with root mean square deviations of 1.07Å for Cα atoms of 455 residues per monomer. Both enzymes are functional dimers and contain a metal triad in each active site. As the superposition of the structures in figure 4.2 shows, there are no major differences in secondary structure content between the two structures.

Figure 4.2 Superposition of the Cα-traces of cold-active shrimp AP (in orange) and human placental AP (in blue): (a), front view, (b) side

view and (c) top view.

Tertiary Structure Both PLAP and SAP contain a metal triad in their active site. In the PLAP structure the binding sites are occupied by two zinc ions and one magnesium ion, with B-factors 20.1, 19.2 and 14.5 Å2 (and a Wilson B-factor of 18.0 Å2). In the SAP structure all three sites were fully occupied by zinc with B-factors 23.6, 23.2 and 27.4 (with a Wilson B-factor of 24.2 Å2). The active site, including metal-ligating residues, is well conserved, except for one conservative substitution: PLAP-S155 is substituted for SAP-T151 (figure 4.3 and appendix A). One important difference in the active site concerns a conserved arginine (PLAP-R166, SAP-R162), which has different conformations in the two enzymes. In PLAP it interacts with the bound phosphate, while in SAP, whose structure does not contain an inhibitor, the arginine has a different conformation. This issue is discussed in detail in chapter 3.

The PLAP structure contains an extra metal binding site, which is occupied by a calcium ion (Mornet 2001). Metal ions often stabilize structures, so PLAP may achieve extra stabilization by an extra metal ion. The loop that binds the fourth metal extends from residue 270 to 285 corresponds to residues 269 to 284 in the SAP model. This loop is located at the tip of the central β-sheet near the cleft of the active site. Several amino acid types are conserved among temperature categories: P270, G271, Y275, D/E276 (among mesophiles and thermophiles); D272, E/D284 (mesophiles/ psychrophiles), H272, I/L277, R/K279, N/E280 (among all categories). This degree of conservation may imply that APs from other species may also bind a metal at this site. SAP shares some of the residues of the metal-binding site, but they have different conformations compared to PLAP. One could speculate whether SAP (and APs from other species) could bind a metal ion at this site. The presence or absence of a fourth metal is either a crystallization artefact in SAP, a crystallization artefact in PLAP, or may be a structural feature restricted to mesophilic (mammalian) APs.

PLAP and SAP both contain two disulfide bridges per monomer, at similar positions. This suggests that the AP family does not use disulfide bridges as a stabilizing factor for mesophiles.

(a) Front view (b) Side view (c) Top view

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8 6 - C H A P T E R 4

In contrast to expectations, SAP contains more ion pairs (defined as non-directional forces between oppositely charged atoms within an distance of 4Å) and hydrogen bonds than PLAP. SAP has 75 ion pairs and 512 potential hydrogen bonds while PLAP has 62 ion pairs and 461 hydrogen bonds. The distribution of hydrogen bond types, whether they are formed between main-chain atoms or side chain atoms, is very similar, but PLAP has additional H-bonds that involve magnesium. In addition, SAP has one salt bridge more than PLAP. The salt bridge between SAP-Asp182 and Arg223 is similar to PLAP Asp185 and Arg227, at the tip of the central β-sheet. The additional salt bridge in SAP is formed between Asp332 and Arg279, in a loop that connects two strands of the central β-sheet. The corresponding residues in PLAP, a lysine and an arginine, have different orientations and do not form a salt bridge.

PLAP contains a putative collagen-binding loop, which extends from residue 400 to 430 (SAP 399-431) and is located in the crown domain. It is not certain if this loop has a function in SAP. Twelve positions in this loop have similar residues in mesophilic and psychrophilic enzymes. Since the crown domain is part of the dimer interface, several residues are located at the dimer interface: eleven for SAP and six for PLAP. SAP, having more residues with higher B-factors in the crown domain, has seven flexible residues in this loop, while PLAP has none (see next subsections).

Figure 4.3 Superposition of the active site residues of

SAP (light grey) and PLAP (dark grey). Most of the

residues superimpose well, except for residues SAP-

His149/PLAP-His153 and SAP-R162/PLAP-R166, which

have different conformations.

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4 . 3 R E S U L T S - 8 7

Quaternary Structure Psychrophilic enzymes are thought to decrease their interdomain interactions in order to gain flexibility. Decreased interdomain interactions have been observed for several cold-active proteins (Kim 1999; Russell 1998b; Smalås 1994). In agreement with these observations, SAP has a somewhat smaller dimer interface than PLAP. PLAP contributes with more residues to the interface, has an increased surface area by about 8 percent and has more polar atoms located at the interface (table 4.6). In contrast, the number of potential hydrogen bonds that can be formed at the interface is larger for SAP. The quaternary structure is not thought to be significantly influenced by the packing in the unit cell, since the functional dimers of SAP and PLAP superimpose very well. The space group of SAP is P43212. The monomers in the asymmetric unit hardly interact with the tip of their wing regions, but the packing of the cell shows crystal contacts via the dimer interface (figure 4.4). PLAP crystallized in space group C2222, with one monomer in the asymmetric unit, and has crystal contacts via the crown domains (figure 4.4). Despite the differences in crystal packing, the functional dimers do not seem to be significantly affected. Figure 4.4 Packing of the unit cells (in Cα traces) of SAP (left) and PLAP (right). The contents per asymmetric unit have the same shade of

grey. The SAP packing is more "open" than suggested in the figure.

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Table 4.6 Properties of the overall structure and the dimer interface

SAP PLAP

Overall structure

Solvent accessible surface area of dimer (Å2) (a) 28446 28923

Volume inside the accessible surface (Å3) (a) 162482 158250

Accessible Surface / Volume ratio 0.18 0.18

Molecular surface area of dimer (Å2) (a) 26619 27031

Volume inside the molecular surface (Å3) (a) 127624 123032

Molecular Surface / Volume ratio 0.21 0.22

Volume atoms (Van der Waals spheres) (a) 83485 81124

Packing density (volume atoms / volume molecular surface) 0.65 0.66

No. cavities per dimer (b) 15 15

Volume of cavities per dimer (Å)3 (b) 225 267

No. ion pairs per monomer (d) 75 62

No. salt bridges per monomer (d) 3 2

No. H-bonds per monomer (c) 512 461

main chain - main chain 279 (54%) 267 (60%)

main chain - side chain 144 (28%) 120 (26%)

side chain - side chain 89 (17%) 74 (16%)

Dimer interface

Nr. residues in interface (e) 84 67

charged atoms at interface (including His) (%) 30 (38) (36%; 45%) 18 (24) (27%; 36%)

polar atoms at interface (%) 35 (42%) 18 (27%)

hydrophobic atoms at interface (%) 20 (24%) 32 (48%)

aromatic atoms at interface (%) 9 (11%) 4 (6%)

Buried surface area (Å2) (f) 3474 4455

Percentage of buried surface area (g) 21% 25%

No. Intersubunit H-bonds (h) 124 84

No. Intersubunit salt bridges 0 0 (a) The accessible surface area, the molecular surface area, the volume inside the molecular surface area and the volume of the

atoms were calculated by GRASP (Nicholls 1991). (b) Voids were calculated with VOIDOO (Kleywegt 1994a), with a probe radius of 1.2Å and a minimum of 5 voxels per cavity). (c) H-bonds were calculated with HBPLUS (McDonald 1994) per monomer, an absence of ligands or water molecules. (d) Ion pairs were calculated with CONTACT (CCP4) (Skarzynski 1988): salt bridges (interactions between charged side chain

atoms from Asp, Glu, Arg and Lys within 4.0Å) were calculated and manually inspected. (e) The dimer interface was calculated with DIMPLOT (Wallace 1995). Residues from the LIGPLOT output file were examined. (f) The buried surface is calculated as twice the molecular surface of the monomer minus the molecular surface of the dimer,

divided by 2: (2*surface dimer-surface monomer)/2. (g) The percentage of the surface of the monomer that is part of the dimer interface is calculated; i.e. buried surface/surface

surface monomer)*100.

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4 . 3 R E S U L T S - 8 9

Figure 4.5

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9 0 - C H A P T E R 4

Figure 4.5 (previous page) Dimer interfaces of SAP and PLAP. The dimer has been “opened” into monomer A and monomer B in order to

visualize the dimer interface. Residues that interact with the other monomer are coloured dark gray. Residues involved in specific

interactions are coloured as described. The top structure is shrimp AP, the bottom structure is human placental AP. In figure (a) the

residues involved in interdimer interactions are shown in gray. In (b) charged residues are coloured. Asp and Glu are red, Arg and Lys are

blue and His is bright blue. In (c) hydrophobic residues Leu, Ile, Val, Met, Phe, Trp, Ala, Pro and Gly are coloured green and in (d) polar

residues Gln, Asn, His Tyr, Ser, Thr and Cys are coloured light blue. This figure can be viewed in full color at the end of the thesis.

Table 4.6 and figure 4.5 show that the residue types participating in intersubunit interactions differ between SAP and PLAP. Overal l Structure: Volume and Surface Properties The accessible surface areas of SAP and PLAP, as calculated with GRASP (Nicholls 1991), are of comparable magnitude (table 4.6). The ratio between these properties has been suggested to be indicative for cold-adaptation, since psychrophilic structures are thought to have a less dense core packing. However, judging from the surface/volume ratio and from the cavity volumes, PLAP seems to be somewhat more compact that SAP, but this difference is not significant. The packing density, defined as the ratio between the volume of the atom's Van der Waals spheres to the space they occupy, of the two proteins is very similar. The surface potential distribution was calculated using GRASP (Nicholls 1991). This program calculates surface potentials at neutral pH. For both enzymes surface potentials were calculated under two conditions, with histidines assigned positively charged and with neutral charge. The electrostatic potentials were plotted onto the surfaces of the enzymes with potentials ranging from -15 (red) to +15 (blue) (in kT/mol=0.6kcal/mol per unit charge).

From a first glance at figure 4.7 it is evident that the surface charge distributions of SAP and PLAP have very different characteristics. With a net charge of -40 (when assigning histidines positively charged) or -72 (when assigning histidines neutral), SAP is highly negatively charged. In both cases the negative charges are distributed almost uniformly on the surface, except for the bottom of the structure and, more importantly, the vicinity of the active site. This effect is more pronounced when histidines are positively charged. The effect of the distribution of charged residues is such that the majority of the surface area is negatively charged, except for a positively charged active site.

PLAP has a net charge of +50 (when assigning histidines positively charged) or +20 (when assigning histidines neutral) and the surface potential distribution is very different from SAP. When histidines are assigned as positively charged, the surface mostly positively charged, especially the crown domain and the bottom of the structure (figure 4.7a,b) with several small negatively charged regions. The nature of the surface potentials changes when histidine is assigned with neutral charge. In that case the surface has more negatively charged areas. The crown domain and the bottom remain mostly positively charged, while the front and the active site groove become more negatively charged.

Overal l Structures: Flexibi l ity Crystallographic temperature factors (B-factors) indicate the degree of atomic mobility. Although one should be careful comparing B-factors between two structures since they depend on several parameters, the comparison between SAP and PLAP may be instructive. Since the two structures have been solved to similar resolution and the crystals were cryo-cooled during data collection, the B-factors may be compared for general trends,

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4 . 3 R E S U L T S - 9 1

though with caution. The SAP structure has a larger Wilson B-factor than the PLAP structure (24.2 Å2 versus 18.0Å2) and displays an overall larger statistical disorder. In order to compare the structures, B-factors of Cα-atoms were "normalized" by division by their overall Wilson B-factors. Inspection of the resulting graphs (appendix B) for residues with higher than average (B/Wilson) value then identified trends and indicated residue stretches with relatively high flexibility.

Although overall B-factors were higher for SAP, the SAP and PLAP structures share structural regions with elevated B-values (figure 4.8). Higher than average (B/Wilson) values were found for the N-terminal helices (1-28), the C-termini (466-476), external loops at the surface (109-124, 169-181, 208-219, 223-228), part of the helix perpendicular to the central β-sheet, loops connecting strands from the main β-sheet to other strands or helices (189-190, 300-303, 341-348) and the crown domain (403-418, 377-385, 470-472). The active site cleft and the central β-sheet were least flexible regions in both structures (figure 4.8). However, the SAP model displays an extension of the commonly found features with longer stretches of the termini (1-30 and 463-465) and more residues of the crown domain (368-372, 376-387, 397-419, 416-429). Longer loops had increased B-factors (209-124, 169-181, 109-126), including the loops between secondary structural elements. Seventeen residues in helices, seven residues in strands and 7 residues on the dimer interface (Asp69, Asp368, Arg369, Tyr398, Pro426, Lys427 and Tyr464) had higher B/Wilson values. In addition to the common features, PLAP has a few residues in the central β-sheet and in loops with relatively high B/W values, amongst which is the helix that runs perpendicular to the central β-sheet. For PLAP, twenty flexible residues are located in helices, six in strands and 6 at the diner interface (Ile1, Ile2, Pro3, Pro65, Tyr367 and Leu448). In addition, residue 477 that is part of the loop that binds the fourth metal has a relatively high B-factor (as well as the corresponding residue in SAP). The zinc ion bound in the third metal-binding site of SAP had a relatively high B/W value, which was not observed for the corresponding magnesium ion PLAP.

Figure 4.6 Illustration of amino acid types at the surface. Hydrophobic residues are coloured green, polar residues are light blue, positively

charged residues are dark blue and negatively charged residues are red. This figure can be viewed in full color at the end of the thesis.

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9 2 - C H A P T E R 4

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Figure 4.7 (previous page) Surface potential representations for SAP and PLAP. SAP is displayed in the upper two figures, PLAP is

displayed in the lower two figures. The calculations were performed under two conditions, with histidine positively charged and with

histidine neutral. The potentials range from –15 (red) to +15 (blue) and have been calculated using GRASP (Nicholls 1991). This figure can

be viewed in full color at the end of the thesis.

Figure 4.8 Trace through Cα atoms coloured by temperature factors divided by the Wilson B-factor (from light grey to dark grey for

increasing B/Wilson value) for SAP (left) and PLAP (right). This figure can be viewed in full color at the end of the thesis.

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4.4 D I SCUSSI ON The AP sequences compared in this study originate from a variety of species, many of which are evolutionary distant. These organisms have been subject to selective pressures or experienced genetic drift, leading to changes in primary sequences that are not necessarily related to cold-adaptation. In principle, one should compare closely related homologues, with representatives from psychrophilic, mesophilic and thermophilic organisms (Sheridan 2000). For this study, a selection was made among the relatively large number of available primary sequences, based on sequence similarity, in order to smooth the effects of genetic drift or selective pressures of different origin. Fortunately the main features of the overall structure and the active site have largely been preserved during evolution, as the crystal structures of E.coli, shrimp and human placental AP have shown. With these restrictions in mind, this study has indicated general trends of amino acid substitutions within the AP family. Residues that are largely conserved among mesophilic sequences ("conserved mesophilic residues", as described in section 4.2.1) have been mutated into other residue types in thermophilic and psychrophilic enzymes. Comparison of "conserved mesophilic residues" and the SAP sequence revealed that many "mesophilic" hydrophobic residues were exchanged for polar residues in SAP and vice versa. With mesophilic APs being more thermostable than SAP, and hydrophobic residues located at the protein core having a stabilizing effect, the substitution pattern may imply that stabilizing core residues in mesophilic APs have been replaced by residues in SAP that have less stabilizing effects. Similar reasoning for polar residues at the surface also implies that stabilizing interactions in mesophilic structures have been replaced by less favorable interactions in cold-active APs. These substitution patterns could be partly responsible for the heat-instability of cold-active APs. It is likely that more residues than the "conserved mesophilic residues" are involved in cold-adaptation. Subtle changes that may have lead to cold-adaptation, e.g. mutations near the active site or dimer interface, cannot be deduced from the "crude" sequence alignments. However, with the few sequences available, the method applied in this study may be adequate to detect general trends within enzyme families.

The conserved mesophilic residues were largely located in a few regions in the structure; in the crown domain, the dimer interface and the wing regions, where they are likely to be responsible for preserving structural elements and intersubunit interactions. They were however hardly present in the central β-sheet, although the overall AP structure is well conserved. The fact that the N-terminal residues are not preserved is not surprising, since this helix is not present in all AP enzymes. In the next subsections, observed trends in the AP sequence comparison have been integrated with the structural characteristics and differences found between the structures of SAP and PLAP. 4 . 4 . 1 S t r u c t u r e S t a b i l i t y The overall structures of mesophilic PLAP and cold-active SAP are very similar and were compared in detail to identify features that may explain the adaptation to cold temperatures. Structural properties shared between the enzymes are not likely to be involved in cold-adaptation: secondary structure content, the metal-binding triad, disulphide bridges, accessible surface area and the compactness of the structure. Characteristics for cold-

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adaptation, an improved catalytic efficiency and heat-instability, possibly accompanied by local flexibility, could partly be explained by the structural comparison. Polar residues, Ion pairs and Hydrogen Bonds Psychrophilic APs generally have lower isoelectric points than their mesophilic and thermophilic counterparts, which is reflected in negative net charges. They also have higher percentages of polar residues and thus the ability to form more hydrogen bonds and ion pairs. This is reflected in the observation that SAP has more hydrogen bonds and ion pairs than PLAP. Since the strengths of electrostatic interactions and hydrogen bonds increase with decreasing temperature, one would expect a thermolabile structure to contain less ion pairs and hydrogen bonds. Indeed, several psychrophilic structures have less ion pairs and hydrogen bonds than their non-psychrophilic homologues (Aghajari 1998; Karlsen 1998; Schroder Leiros 2000), although an increase in these interaction types has been observed as well (Russell 1998b). The fact that SAP contains more of H-bonds and ion pairs than PLAP suggests that the observed heat-instability of cold-active AP enzymes is not caused by a decrease in ion pairs or hydrogen bonds. Most of the polar residues of SAP were located at the solvent-accessible surface, in particular negatively charged residues. Charged residues on the surface have been attributed to higher stability (Aghajari 1998), but in the AP family, charged residues on the surface may not be related to structure stability, but to optimization of the surface charge distribution (section 4.3).

The mean percentage of arginines and the ratio R/(R+K) steadily decreases from thermophilic to psychrophilic APs. The same trend has been observed for more enzymes (Aghajari 1998; Kim 1999). A low ratio has been suggested as an indicator for cold-adaptation in primary sequences, and in the case of the alkaline phosphatase family, the indicator follows the predicted trend. The two AP structures do not show a special (re)distribution of the arginines.

Hydrophobic Residues Buried hydrophobic residues contribute to the stability of protein structures. Psychrophilic APs have significantly lower percentages of hydrophobic residues than their mesophilic and thermophilic counterparts. On top of this, the strengths of the hydrophobic effect diminish as the temperature decreases. So in principle, psychrophilic APs have less hydrophobic residues to stabilize their cores. This is illustrated by SAP having less hydrophobic residues in the interior of the structure than PLAP. In addition, when comparing the "conserved mesophilic residues" to the corresponding residues in SAP, many of the mesophilic hydrophobic residues appeared to be replaced by polar amino acids in SAP. Considering the fact that hydrophobic residues have a stabilizing effect when buried, while polar residues have a stabilizing effect when exposed, the exchange of these residue types must affect structure stability. Assuming that mesophilic enzymes have the more stable structures and the conserved stabilizing interactions are exchanged for destabilizing interactions, it is likely that the psychrophilic structures become less stable upon these substitutions. Altogether, the decrease of potentially stabilizing hydrophobic residues must be partly responsible for the heat-instability of psychrophilic APs. Glycine and Proline Residues Psychrophilic APs have lower percentages of proline and glycine residues than their warm-blooded homologues. Some comparative studies have stated that the locations rather than the percentages of glycines and prolines are important (Aghajari 1998; Smalås 1994). When prolines are located in helices they have a destabilizing effect, since they break at least two hydrogen bonds, unless they are situated at the N-caps, where they induce helix formation. Since SAP and PLAP have the same number of prolines that are located in

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helices, their structural stability is not expected to be influenced. However, PLAP has significantly more prolines located in loops than SAP. This generally is a stabilizing effect because prolines have restricted conformational freedom. Intersubunit Interactions It seems paradoxical that SAP has more interdimer interactions (figure 4.5), which may stabilize the tertiary and quaternary structure, while this enzyme is heat-labile. The observation that PLAP has less intersubunit interactions than SAP can be explained by PLAPs allosteric character, which needs spatial rearrangements. However, dissociated subunits of SAP have been reported to retain enzyme activity (Olsen 1991). Based on insertions in the primary sequence of vibrio AP (VAP) compared to ECAP, VAP is thought to be monomeric as well, since these insertions could protrude the dimer interface (Asgeirsson 2001). When individual subunits are catalytically active, the dimer interface may be less important. For cod AP, flexibility at the interface and perhaps around the active site, have been proposed to enhance the catalytic efficiency, possibly by lowering the affinity for inorganic phosphate (Ásgeirsson 2000), although this has not been experimentally proven. Considering the differences between the structure stability and activity of AP dimers and subunits from different species, it is very likely that the dimer interface is species-dependent and is not a feature related to thermal adaptation. VAP has been suggested to be functional as a monomer or multimer ( sgeirsson 2001). Comparison and alignment of the primary sequences of VAP and SAP showed several inserts for VAP, the most important of which are found between residues 388 and 389 (using SAP numbering): 9 residues, between 399 and 400: 21 residues, and between 348 and 439: 25 residues. The first insert would not affect the oligomerization of the enzyme, since this extension is located at a solvent-exposed part of the crown domain. The second insert, also located at the crown domain, is close to the dimer interface, which may prevent dimer formation. The last insert is at the center of the extended β-sheet, close to the dimer interface and may therefore influence dimer formation. 4 . 4 . 2 D i s o r d e r a n d F l e x i b i l i t y In principle crystallographic temperature (B-) factors indicate the dynamic mobility for atoms, but one should be careful when relating B-factors to local dynamic behavior in a crystal structure. B-factors are dependent on data quality, resolution, packing, disorder in the crystal lattice, solvent content, temperature at which data sets have been collected and the refinement procedure. They may however be useful for indicating local variations between structures (Russell 1998b).

PLAP and SAP share common features with respect to disorder (indicated by elevated B/W values), although the flexible stretches are slightly prolonged in the SAP structure, especially in the crown domain and in loops connecting strands and helices. These observations can however not directly explain an improvement in catalytic efficiency, since they do not seem to influence the catalytic site. The heat-instability might be slightly affected, since the crown domain, with somewhat elevated disorder, is part of the dimer interface.

The zinc ion in the M3 site in SAP had a relatively high B/W value, while the magnesium ion in PLAP is tightly bound. In contrast to SAP, where anomalous data confirmed the presence of zinc in all sites, the identities of the metal ions metals in PLAP were not checked. However, the conformations of the ligating residues of the M3 site in PLAP indicate the presence of magnesium. More specifically, H153 does not

Á

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coordinate the magnesium ion, while the corresponding histidine is SAP does coordinate the zinc ion. Although this observation indicates the presence of magnesium in PLAP, one cannot rule out the possibility of mixed occupation by magnesium and zinc. If both metals were present while only magnesium was modeled, one would expect either a higher (than actual) magnesium occupancy (the electron density from zinc being "filled up" by extra magnesium) or, with similar reasoning, a lower B-factor. One cannot be sure whether the magnesium ion in PLAP genuinely is tightly bound, which is indicated by the low B-factors of the magnesium ion and the ligating residues, or that there is mixed occupancy of magnesium and zinc.

Glycines located in loops or hinge regions may introduce extra flexibility. However, PLAP has more glycines in helices and coils than SAP, which suggests that glycine residues are not likely to be responsible for the flexible regions in SAP.

Several techniques can provide information about the flexibility or disorder in protein structures, such as hydrogen/deuterium exchange experiments (Zavodszky 1998), nuclear magnetic resonance (NMR) spectroscopy (Palmer 2001) and molecular dynamics (MD) simulations. MD simulations have shown that cold-active trypsin has a higher flexibility near the active site, which may lower the activation energy for ligand binding and catalysis (Brandsdal 1999). Currently MD simulations are being performed on SAP. These studies may provide answers with respect to the question whether or not SAP profits from an increased efficiency due to (local) flexibility. 4 . 4 . 3 C a t a l y t i c E f f i c i e n c y Enzymes can improve their catalytic efficiency kcat/KM by increasing the turnover number kcat or by improving substrate affinity (decreasing the value of KM). SAP has probably evolved to increase the turnover number by optimizing the surface charge distribution. The excess of negative charges (Nilsen 2001) is distributed such that the negatively charged substrate is attracted towards the positively charged active site (figure 4.7). Electrostatic interactions between SAP and ligands have already been indicated by Olsen et al (Olsen 1991), who suggested that a negatively charged ligand at pH 5.7 bound to a specific positively charged site. This implies that SAP still attracts substrates at lower pH, although the effect may be less effective. Ligand binding experiments or electrostatic surface calculations at different pH and ionic strengths could be performed in order to illustrate this effect. Diffusion of substrates to enzymes influenced by an electric field has been demonstrated (Sharp 1987) and optimization of surface potentials has been suggested before as a strategy for cold-adaptation, as similar effect has been observed for some cold-adapted proteins (Gorfe 2000; Kim 1999; Russell 1998b; Smalås 1994).

The surface charge distribution is very different for the two enzymes, and may be an important factor for cold-adaptation. The surface of SAP is highly negatively charged, except for one positively charged patch per monomer, located around the metal triad (figure 4.7). The distribution of positive and negative charges may guide the negatively charged substrate towards the positively charged active site. Considering the fact that APs have an alkaline pH optimum for catalysis, it is interesting that the positively charged region in the vicinity of the active site is more pronounced when histidines are assigned positively charged, which is the case in an acidic environment.

The attractive effect on negatively charged compounds, which guides the substrate towards the active site, may well increase the efficiency of the enzyme, but it must also apply to the product and thus hinder its departure. One may speculate that the attractive force is weaker in an alkaline environment, but still sufficient to attract substrates and not hindering departure of the product as much. The net effect would then be an

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enhancement of the overall efficiency of the enzyme. The potential surface of PLAP is very different and does not show the marked charge differentiation observed for SAP.

It would be interesting to calculate the surface potentials at alkaline pH, since APs have an alkaline pH optimum. Unfortunately GRASP is not able to do these calculations and more advanced software is needed. On the other hand, the physiological pH must be close to 7, so the calculation described in this section resembles the physiological environment more closely.

Although the active sites of PLAP and SAP are practically identical apart from a conserved substitution, changes in the vicinity of the active site may account for reduced phosphate affinity. The chemical environment of active site arginine R162 is such that it prefers an "upward" conformation, in which it does not interact with the substrate or ligand. A local hydrogen-bonding network keeps the arginine in this conformation. The corresponding arginine in PLAP has a "downward" conformation and interacts with the bound substrate. These conformations are described and illustrated in chapter3. In an alkaline environment, hydrolysis of the noncovalent phosphoenzyme intermediate complex is the rate-limiting step in the catalytic cycle. If phosphate would be less tightly bound, this may help its release and therewith improve the catalytic efficiency. One cay only speculate as to whether the phosphate is bound less effectively (with lower affinity) and if this effect increases the catalytic efficiency kcat/KM. Although the possibility remains that this characteristic is a crystallization artifact, this is not very likely. Crystallization experiments of SAP with various conditions and ligands (described in chapter 3) have shown that although R162 can adopt both conformations, it predominantly occupies the conformation in which does not interact with the ligand. It is uncertain whether this feature is specifically to SAP or if it is a general feature of cold-adapted APs to improve catalytic efficiencies. In fact, several cold-active APs are known to have reduced or altered interactions with substrates or inhibitors (Ásgeirsson 1995; Hauksson 2000).

Some psychrophilic enzymes have been observed with a better active site accessibility compared to their temperate homologues (Russell 1998b). The active sites entries of PLAP and SAP are very similar (figure 2.17), so increased substrate accessibility is not likely to contribute to an improved higher efficiency. Temperature Opt imum The temperature optimum of activity for an enzyme is not necessarily related to the physiological environment of the organism. Enzymes are often found to have optimal temperatures of activity that are ten to twenty degrees higher than the optimal growth temperature of the parent organism (Marshall 1997). The temperature optima of APs from several species have been found to be the same, irrespective of the respective thermostability (Lustig 1971), although more recently the temperature optima of TAB5 and HK47 were found to be 25ºC (Rina 2000). The temperature optimum for both SAP and PLAP is 37ºC. Apparently the optimum temperature for activity does not play a role in the AP family. The kinetics at physiological conditions are more important, since inorganic phosphate, a regulator of extracellular AP activity, is likely to inhibit most AP enzymes (Coburn 1998).

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4.5 CONCLUSIONS The alkaline phosphatase family only uses a selected array of structural adjustments for cold-adaptation. In order to improve catalytic efficiency, shrimp alkaline phosphatase has optimized surface potentials in order to attract the substrate. Ligand binding experiments and surface potential calculations at various pH and ionic strengths could be performed to further characterize the effect of the surface potential on the catalytic efficiency. Additionally, an alternative conformation of active site arginine R162, which does not interact with the bound ligand like the corresponding arginine in PLAP, may facilitate the release of product, thereby enhancing the catalytic efficiency. Heat-instability is largely explained by a reduction in hydrophobic residues in the protein core and a reduced number of stabilizing arginines. In addition, PLAP is stabilized by an extra bound metal and additional prolines in loop regions, although SAP has more hydrogen bonds, ion pairs and polar residues. Flexible regions at the dimer interface of the crown domain may also contribute to heat lability. In order to study the heat-instability and possible flexible regions of SAP, additional experiments must be performed.

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