Role of C-terminal B-chain residues in insulin assembly:the structure of hexameric LysB28ProB29-human insulin

Ewa Ciszak l , John M Beals2, Bruce H Frank2, Jeffrey C Baker 2,

Nancy D Carter 2 and G David Smith1 ,3*1Hauptman-Woodward Medical Research Institute, Inc., 73 High Street, Buffalo, NY 14203, USA, 2Eli Lilly Research Laboratory,

Indianapolis, IN 46285, USA and 3Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263, USA

Background: LysB28ProB29-human insulin (HumalogT ),a fully potent insulin analog in which the prolyl, lysylsequence at the C-terminal end of the B-chain is inverted,exhibits a decreased association of monomers to dimersleading to rapid in vivo absorption. This provides impor-tant benefits for the insulin-requiring diabetic. In spite ofits monomeric nature, LysB28ProB29-human insulin canexist as a discrete hexameric structure in the presence ofboth zinc and phenol. Studies of the crystal structure ofLysB28proB29-human insulin in a hexameric complex wereinitiated to gain a molecular understanding of the effect ofthe sequence inversion on the analog's self-associationproperties and, consequently, its in vivo efficacy.Results: Under the conditions reported, LysB28ProB29-human insulin crystallized as a T3Rf hexamer that is iso-morphous with the uncomplexed T3 R3 native human

insulin hexamer previously known as '4Zn insulin'. Thethree-dimensional structure of the T3R

f hexamer wasdetermined by X-ray crystallographic methods to a reso-lution of 2.3 A. The prolyl, lysyl sequence inversion leadsto local conformational changes at the C termini of theB-chains which eliminate two critical hydrophobic inter-actions and weaken two terminal 3-sheet hydrogen bondsthat stabilize the dimer.Conclusions: The loss of these native dimer interactionsweakens the hexameric LysB28ProB29-human insulincomplex formed in the presence of phenolic ligands.Thus, it is hypothesized that the diffusion of the phenolicligands from the site of injection results in the dissociationof hexamers directly to monomers, thereby maintainingthe rapid time-action of the monomeric analog in spite ofthe hexameric conformation in therapeutic formulations.

Structure 15 June 1995, 3:61 5-622Key words: dimer destabilization, hexameric insulin, insulin analog, rapid time-action, sequence inversion

IntroductionThe human insulin molecule consists of a 21 amino acidA-chain and a 30 amino acid B-chain, linked by twodisulfide bonds. The association of insulin monomers todimers and larger aggregates is a complex, dynamic equi-librium and is a function of pH, ionic strength and pro-tein concentration. Insulin is capable of binding variousdivalent metal ions although it is synthesized and stored inthe pancreas as a Zn2 +-containing hexamer. X-ray cryst-allographic studies of hexameric insulin have shown theinsulin molecule to be very flexible, as discrete conforma-tional states have been observed for the N terminus of theB-chain [1-4]. It has been suggested that conformationalchanges of the N terminus can be related to loss of Zn2+

and the subsequent dissociation of the hexamers to thebiologically relevant monomers [2]. Moreover, the C ter-minus of the B-chain is believed to possess inherent flexi-bility, which is critical for self-association of insulinmolecules and the binding of insulin to its receptor [5-7],as evidenced by the loss of activity of the single-chainB29-A1 peptide-linked insulin. The dimer is stabilized byhydrogen bonds in a short antiparallel 3-sheet consistingof the B-chain C-terminal residues PheB2 4-TyrB 26. Theimportance of the C terminus of the B-chain forself-association is shown by removal of residues B26-B30,despentapeptide insulin, which cannot dimerize through

this region of the molecule [8]. Although removal of B29and B30 does not significantly reduce association, theremoval or replacement of ProB2 8 does alter these proper-ties, which strongly suggests that ProB2 8 is critical todimer stabilization and hence hexamer formation [9].

LysB28ProB2 9-human insulin, an insulin analog created byan amino acid inversion of ProB28 to lysine and LysB 29 toproline, has been used to investigate further the role ofthe C-terminal B-chain residues in self-association. Thisinversion has been found to diminish the dimerizationconstant by a factor of 300, and hence hexamer forma-tion [9]. However, in spite of its greatly diminished self-association properties, this monomeric analog can bestabilized in discrete hexameric structures by the additionof both zinc and phenol. Here we report the structure ofa hexameric complex generated in the presence of zincand phenol and identify specific changes in intermolecu-lar interactions in the dimer that are responsible for themonomeric properties of this analog. In addition, thesestructural results also suggest an explanation for the abil-ity of LysB28proB29-human insulin formulations (contain-ing both zinc and phenolic preservatives) to retain thetime-action of a monomeric analog, that is, rapid onsetof activity, in spite of the hexameric association state inthe pharmaceutical preparation.

*Corresponding author.

© Current Biology Ltd ISSN 0969-2126 615

616 Structure 1995, Vol 3 No 6

Results and discussionMonomer nomenclatureLysB28ProB29-human insulin crystallizes as a hexamer,illustrated in Figure 1, that contains two zinc ions andthree phenol molecules. The hexamer is composed ofthree insulin dimers arranged about a central crystallo-graphic threefold axis. The two independent monomerswhich comprise each dimer differ significantly in confor-mation, especially at the N termini of their B-chains(Fig. 2). The differences in conformation at the first eightresidues of the N termini of the B-chains have beendescribed by the T and R nomenclature, where T and Rrefer to an extended or an a-helical conformation,respectively [10]. Residues PheB1 l-GlyB8-l (where the 1or 2 in the decimal portion of the chain name andresidue number refers to the T-state monomer or theR-state monomer, respectively) of the zinc complexof LysB2 8 ProB2 9-human insulin shown in Figure 2a arevery similar to those observed in the 2Zn insulin (T6)

Fig. 1. The LysB28ProB29 -human insulin hexamer viewed alongthe threefold axis. Rf-state monomers are colored red, T-statemonomers green, phenol molecules magenta, disulfide bondsyellow and zinc red. As the hexamer is viewed along the three-fold axis, only one zinc ion is visible. (Drawn with SETOR [201.)

structure [1]. The R-state conformation observed in R 6insulin hexamers [4,11], in which residues B1-B8 area-helical, is illustrated in Figure 2b. Unlike the pureR-state conformation, in which there is a continuousat-helix from B1 to B19, a continuous or-helix is presentonly for residues B4.2 to B19.2 in LysB28ProB2 9-humaninsulin. This 'frayed' a-helical conformation is defined asthe Rf-state in order to distinguish it from that observedin R hexamers, and is shown in Figure 2c. TheLys12LProB29-human insulin hexamer is therefore classi-fied as T3R3f. However, this R f conformational state andthe resulting T3R3f hexamer are not unique to this insulinanalog. An analogous conformation has been observed inthe hexameric insulin previously referred to as 4Zninsulin [3], in the insulin-p-hydroxyacetanilide complex[12], and in the insulin-p-hydroxybenzamide complex[13], all of which are isostructural with the LysB28proB29-

human insulin analog. The Rf nomenclature had notbeen explicitly defined at the time these structures werereported, however, and they were classified as simply R.In the Rf-conformational state the ag-helix is disrupted atAsnB3 2 by a positive angle of 111 ° . The AsnB32 sidechain is in a position where it can accept hydrogen bondsfrom the main-chain nitrogen atoms of both HisB5 2 andLeuB6 2, and thus act as a surrogate for the first tworesidues of the B-chain in the R conformational state.Therefore, the extended conformation of the first threeresidues is energetically equivalent to the a-helical con-formation, as the number of hydrogen bonds remainsunchanged.

Hexamer-hexamer interactionsEach T3R3f LysB28proB29-human insulin hexamer can bethought of as constructed from two threefold symmetrictrimers, T3 and R f, which are assembled around thethreefold axis. The surface of each trimer is differentbecause of the conformational differences of the N-ter-minal B-chain residues. The two Zn2 + ions (Znl andZn2) lie on the threefold axis and each is coordinated bythree symmetry-related His B 1° residues of the T- orRf-state monomers. Znl lies at the bottom of a shallowdepression on the surface of the T3 trimer, where it isoctahedrally coordinated by three HisB 1i° ' residues andthree water molecules (Znl-Ne2, 2.15 A; Znl-OW1,3.53 A). In the R f trimer, Zn2 is also bound by threesymmetry-related HisBI10 2 residues (2.10 A), but as aresult of the presence of the a-helix resulting from theT--R f transition (due to the presence of chloride ion or

Fig. 2. Conformation of the B-chainsin the (a) T-state, (b) R-state and (c)RL-state monomers.

Role of B-chain in insulin assembly Ciszak et al. 617

Finally, the alignment of the hexamers along the c-axisresults in infinite chains of hexamers in which access toeither the T or R f Zn2 +-binding site is blocked by theadjacent hexamer.

Fig. 3. Ribbon representation of the inter-hexamer interactions atthe T and Rf surfaces. T-state monomers and the side chain ofHis B5 are illustrated in green. The Rf-state monomers and theside chains of PheB 1 2, Val B2 2, and Leu B6 2 are illustrated in red.Also illustrated are the two independent zinc ions, colored purple.

phenolic derivatives), there is insufficient space for octa-hedral coordination. Consequently, the coordination atthis site is restricted to tetrahedral, the fourth ligandbeing a chloride ion, which is also located on the three-fold axis at a distance of 2.31 A from Zn2.

As hydrogen bond donation near the N terminus of theR or Rf a-helical segments is satisfied for either con-formation, the selection of the R f conformation by bothhuman insulin and LysB28proB2 9-human insulin can bebest explained on the basis of the packing of the hexa-mers along the c-axis. The generation of a pure R-stateconformation, in which residues PheB I 2 to AsnB3 2 area-helical, would result in unacceptable interactionsbetween the T and R trimers which could' only berelieved by increasing the length of the c-axis and henceincreasing the inter-hexamer separation. This increasewould in turn expose the side chain of PheBI to thesolvent as well as increase the solvent content of thecrystals. In the R f conformation, however, residuesPheBl 2 to Asn B3 2 project away from the R f trimersurface where, together with their threefold symmetry-related counterparts, they form a 12 A deep channel.The three PheBl' 2 side chains nestle into the T3 surfaceof a translationally (c-axis) related hexamer and are nearlyparallel to the T-state HisB5.1 side chains (Fig. 3). Theseinter-hexamer interactions between the T and R f

3 3trimers serve to shield the PheB1' 2 and ValB 2 2 side chainsfrom the surrounding solvent and consequently facilitateefficient packing of the T3 and R3f surfaces against eachother. Although the PheB1'2 and ValB2 2 side-chainconformations are stabilized by hydrophobic interactions,no directed interactions (such as hydrogen bonds) stabi-lize the N terminus of the B-chain in the Rf-state mono-mer. Consequently, some conformational flexibilityresults in this region, as shown by the Ca displacementsobserved in a comparison of the TR f LysB28ProB 2 9-

human insulin dimer and the TR f 'phenol-less' humaninsulin dimer (Fig. 4a).

TRf dimerAs stated above, Figure 4a shows a comparison of theconformation of the backbone atoms of the TR f

LysB2 8proB 29-human insulin dimer with that of the TR f

human insulin dimer. Their overall similarity is reflectedin average and root mean square (rms) displacements of0.25 A and 0.28 A, respectively, minimizing the displace-ments of the main-chain atoms of All-A19 andB11-B19. A comparison of the three disulfide bridges ineach monomer of the LysB28proB29 -human insulindimer with those observed in the human insulin TRfdimer reveals no significant differences.

Even though the sequence inversion at B28 and B29 haslittle effect upon the overall conformation of the dimer,there are five regions in which significant differences areobserved: ThrA8 2-IleA 10.2, PheB 2 -AsnB 3 2, GlyB 2° '2_GlyB23 2 and ThrB27.2-ThrB30 2 in the Rf-state monomerand ThrB 27 l-ThrB3' (1 in the T-state monomer. Minordisplacements (less than 1.0 A) in the ThrA82-IleA0O2

sequence have been observed in other T3R3f insulinhexamers [3,12,13]; to date, a satisfactory explanation forthese differences has not yet been made, nor is one obvi-ous in the present work. The origin of the displacementsat PheBl2-AsnB3 2 (approximately 2 A) have been dis-cussed above, under 'Hexamer-hexamer interactions'.Differences in the B27-B30 region (2-4 A) wereexpected, as this sequence contains the inversion site.The displacements observed in the B20-B23 region(1-2 A) are due to the loss of its interactions with residueProB2 8. Moreover, both the B20-B23 and B27-B30 seg-ments lie on the hexamer surface and are responsible forhydrophobic interactions that contribute to the stabiliza-tion of the TR f dimer.

A superposition of the T- and Rf-state monomers ofLysB28 ProB29-human insulin (Fig. 4b), minimizing thedisplacements of backbone atoms in residues All-A19and B11-B 19, resulted in an average displacement of0.54 A and an rms displacement of 0.61 A. In Figure 4b,regions PheBl-LeuB6 and ProB29-ThrB30 can easily beidentified as having the most significant displacements.The largest conformational difference between the twoindependent monomers in LysB28proB29-human insulinis associated with the T-4R f transition, in other words,the extension of the at-helix at the N terminus of theB-chain and the 20 ° rotation of the initial seven residuesof the A-chain.

In the human insulin TR f dimer, the B-chain a-helicesterminate at GlyB20 and a hydrogen bond associated withthe a-helix is observed between the B20 amino nitrogenand the carbonyl oxygen of B16. Residues B20-B23form a type III 13-turn and have nearly identical confor-mations in both monomers. In contrast, the B20-B23

618 Structure 1995, Vol 3 No 6

Fig. 4. A comparison of the displace-ments of the Ca atoms of (a) the TRf

dimer of LysB28ProB 29-human insulinwith that of the TRf phenol-less humaninsulin dimer, and (b) the LvsB28ProB 29-

human insulin T-state monomer withthp Rf-sttep monnmPr

loops in LysB28 ProB2 9-human insulin have significantlydifferent conformations in each of the monomers. In theT-state monomer, this loop adopts a distorted type III,3-turn, but in the Rf state, the turn is severely distortedand no hydrogen bond exists between GlyB2 (' 2 andGlyB23 2 (Table 1). Although the terminal B20-B16hydrogen bond in the -helix exists in the T-statemonomer, this hydrogen bond is not present in theRf-state monomer, because of the distortions in the

B20-B23 loop, which may be due to the proximity ofatoms in an adjacent hexamer or to the formation of ahydrogen bond between No of LysB 28 '1 and the carbonyloxygen of GluB 2 L 2 (Table 1).

Although the sequence inversion at B28 and B29 has lit-tle effect upon the overall conformation of the dimer, itstrongly affects the hydrophobic and hydrophilic interac-tions between T and Rf monomers. Dimer stabilizationin T 3Rf native insulin is achieved in part by hydrophobicinteractions as well as by surface complementaritybetween the prolyl rings of each monomer and theB20-B23 loops in the other monomer (Fig. 5a). Inhuman insulin, the side chain of Pro B2 8 nestles into theB20-B23 loop of the adjacent monomer, where it makesnumerous hydrophobic interactions with main-chainatoms of GlyB2(, GluB2 1 and GlyB23 . In LysB28proB2 9-human insulin, however, the sequence inversion displacesproline towards the C terminus, making hydrophobicinteractions between proline and the B20-B23 loopimpossible (Fig. 5b). Even if the B-chains made turns atthis point as they do in human insulin, the LysB28 sidechain is too long to substitute for the prolyl ring and pro-vide the corresponding hydrophobic interactions withthe B20-B23 loop of the other monomer. It is interest-ing to note that the thermal parameters of the T-state

Table 1. Comparison of torsion angles and hydrogen-bonddistances in the B20-B23 loops of LysB28ProB29-human insulinwith those of human insulin.

LysB28proB 29 Human

T-state Rf-state T-state Rf-state

Torsion angles (°)~21 -28 -84 -65 -53qs21 -37 -15 -17 -32~22 -79 -11 6 -63 -69tJ22 -30 -20 -42 -23

Hydrogen-bond distances (A)GlyB20 O-GlyB 23 N 2.72 3.98 3.09 3.19TyrB16 O-GlyB20 N 2.98 3.56 3.13 3.06LysB28 1 No-GluB2 1' 2 0 2.55 -

I -l '- "'t t ....

Role of B-chain in insulin assembly Ciszak et al. 619

Fig. 6. A superposition of the sequence TyrB2 6.l-ThrB30l in

human insulin (red) as compared with the same fragment inLysB28ProB29-human insulin (colored according to atom type).

Fig. 5. The interface between the C terminus of. the T-statemonomer (B26-B30) and the loop of the Rf-state monomer(B19-B24) in (a) human insulin where residue label D28PROrefers to ProB28.2 while B20GLY and B23GLY refer to GlyB20 1

and GlyB231, respectively; and (b) LysB28ProB2 9-human insulin

where residue label B228LYS refers to LysB28.2 while B120GLYand B123GLY refer to GlyB201 and Gly B23'1, respectively.

loop and the Rf-state C terminus are comparable inLysB28proB29-human insulin and human insulin, but thatthere is a substantial increase in the thermal parameters ofLysB2 8proB2 9-human insulin (12 A2) for the Rf-stateloop and the T-state C terminus.

The loss of the hydrophobic interactions with theB20-B23 loop also contributes to the weakening of somehydrogen bonds in the 1-pleated sheet region (B24-B26)that are critical for stabilizing the dimer. While the innertwo hydrogen bonds in the -pleated sheet are com-parable to those observed in human insulin, the outertwo hydrogen bonds have lengthened by an average of0.35 A (Table 2).

Finally, the displacement of proline eliminates theintramolecular packing of the ProB2 8 ring against the sidechain of TyrB26 in the monomer (Fig. 6). The elimination

of this interaction reduces the conformational stability ofthe C-terminal residues of both monomers and permitsadditional flexibility of the C-terminal residues inLysB28ProB 29-human insulin.

Phenol-binding sitesLysB28 ProB2 9-human insulin binds only three phenolmolecules, unlike human insulin, which under similarcrystallizing conditions binds six phenol molecules in anR 6 state. Phenol molecules are bound in ellipticallyshaped binding sites located on the interfaces between Rfmonomers through a pair of hydrogen bonds from thephenol hydroxyl group to the carbonyl oxygen ofCysA6 2 (2.66 A) and the nitrogen of CysAl 2 (3.20 A).This binding pattern is observed in the structures of allreported human insulin-phenol complexes [4,11], andalso in complexes with larger phenolic derivatives such asp-hydroxyacetanilide [12] or p-hydroxybenzamide [13].In LysB28proB29-human insulin, that portion of thebinding cavity that continues beyond the phenol mol-ecule towards the center of the hexamer is occupied bytwo water molecules, OW3 and OW33, which formhydrogen bonds to each other (2.77 A) as well as to thecarboxyl group of GluB 13 1 (OW33-Oe2, 3.41 A) andthe carbonyl oxygen of HisB10 2 (OW3-O, 2.95 A).Both of these water molecules have counterparts in thestructure of the phenol-less T3R 3f human insulinhexamer [3].

Table 2. Comparison of the -pleated sheet hydrogen-bondlengths (A) in the TRf dimer of LysB28ProB29-human insulinwith those of human insulin.

Monomer I Monomer I

Residue Atom Residue Atom LysB28ProB29 Human

TyrB 26 1 O PheB24.2 N 3.23 () 2.92 (A)TyrB26.1 N PheB24.2 O 2.89 (A) 2.93 (A)PheB 24.1 O TyrB26.2 N 2.72 () 2.80 (A)Phe B24.1 N TyrB26 2 O 3.25 (A) 2.89 (A)

620 Structure 1995, Vol 3 No 6

Fig. 7. Schematic comparison of thehypothesized dissociation of LysB28 roB29-human insulin and insulin in subcuta-neous tissue. The insulin hexamers in theformulation are represented as a dimer oftrimers (where a triangle represents aninsulin monomer), with one trimer inlight blue and the second trimer high-lighted as purple. Each trimer coordinatesa zinc ion (orange sphere); as the hexa-mer is viewed along the threefold axis,only one zinc ion is visible. The stippledand unfilled monomers represent thepresence and absence of bound phenolicpreservative, respectively. The free phe-nolic preservative is represented as agreen O. The subcutaneous tissue is rep-resented as pink boxes. The hexamer dis-sociates in the subcutaneous tissue and isthen absorbed (unpaired triangles)through the capillary membranes in thesubcutaneous tissue.

Rapid time-action from hexameric preparations ofmonomeric analogsMany therapeutic preparations of human insulin con-tain Zn2+ and a phenolic derivative as a preservative.These preparations are necessarily hexameric (due tothe presence of zinc ion) and current models suggestthat the rate-limiting step in the absorption of insulinfrom a subcutaneous injection site is the dissociation ofhexamers to the biologically relevant monomers [14].Clinical studies have verified that monomeric prepara-tions of LysB2 8ProB29-human insulin [15] or othermonomeric analogs [14] are more rapidly absorbed intothe blood than hexameric human insulin preparations,supporting the hypothesis that the rate-limiting step ininsulin absorption is the dissociation of hexamers todimers and monomers.

However, just as in the case of human insulin therapeuticpreparations, monomeric analogs require formulationsthat yield both suitable chemical and physical stability.Stabilization of LysB2 8ProB2 9 -human insulin is achievedby the addition of zinc and phenol, which act to inducethe otherwise monomeric analog to associate into discretezinc-containing hexameric complexes under pharma-ceutically useful concentrations (Bakaysa, D.L., et al., &Radziuk, J., personal communication). We would empha-size that both phenol and zinc are necessary to form dis-crete LysB28ProB29-human insulin hexamers. Surprisingly,in spite of the formation of this hexameric state, recentclinical data show that the time-action of LysB28proB2 9-

human insulin is not significantly affected [15].

The retention of the rapid time-action of this analog canbe explained in part by comparing the subtle structuraldifferences that are observed between the T3R3f crystalstructures of LysB28proB29-human insulin and nativehuman insulin. In the latter structure, two sets of interac-tions involving residues B20-B23 of each monomercontribute to the stabilization of the dimer: firstly,

hydrophobic interactions between each ProB2 8 and theB20-B23 loop of the other monomer in the dimer, andsecondly, four hydrogen bonds across the 3-pleated sheet.The sequence inversion at B28-B29 results in poorersurface complementarity, which disrupts the hydro-phobic stabilizing effect of ProB28 and contributes to alengthening of the hydrogen bonds at each end of the3-pleated sheet by approximately 0.35 A. Consequently,

these weakened dimer-stabilizing interactions coupledwith the requirement of both phenolic preservative andzinc for the stabilization of the LysB2 8 ProB2 9-humaninsulin hexamer explain how a monomeric analog for-mulated as a hexamer can retain its rapid time-action,that is, the ability to readily dissociate into a rapidlyabsorbed monomeric compound (Fig. 7).

We hypothesize that after a subcutaneous injection theexcess, unbound phenolic preservatives are rapidlyabsorbed into the surrounding tissue. The loss of thisexcess preservative, coupled with the dynamic structuralchanges at the N terminus of the B-chain (Rf-T), shiftsthe phenol-binding equilibrium, destabilizing theLysB28ProB29-human insulin hexamer. The hexamersthen readily dissociate into monomers as a result of theweakened dimer-stabilizing interactions. The dissociationof the LysB28proB29-human insulin hexamers differs fromthat of human insulin (because of their differential stabil-ity in the absence of phenol) such that the diffusion ofphenolic preservatives from a human insulin hexamerwould not alter the association state of the protein, butonly the conformational state (R-T). Thus, in the caseof native human insulin, elimination of the zinc and dilu-tion of the insulin is required to dissociate the hexamerfirst to dimers and subsequently to monomers.

Finally, the T and R f conformations, which fall withinthe envelope of structures described for the insulin mol-ecule, show that the flexibility of the insulin molecule isretained in this analog. This is important not only from a

Role of'B-chain in insulin assembly Ciszak et al. 621

structural perspective but also a biological perspective asinsulin's flexibility has been related to its activity [6].Based on this crystal structure, it can be seen that none ofthe residues implicated in receptor binding undergoeschanges in conformation and consequently the analogmaintains full biological potency, that is, the ability toelicit a full biochemical response [1].

Biological implicationsThe dissociation of zinc-insulin hexamers intomonomers is hypothesized to be the rate-limitingstep in the absorption of insulin from a subcuta-neous injection. Thus, to accelerate this absorp-tion process, a monomeric insulin analog wasdeveloped by inverting the prolyl, lysyl sequenceat residues 28 and 29 of the human insulinB-chain, yielding LysB2 ProB29-human insulin.This insulin analog displays monomeric propertiesunder in vivo conditions as a result of a decreasedpropensity of the analog to dimerize. However, inspite of a -300-fold decrease in the dimerizationconstant, LysB28 ProB29-human insulin can stillassociate into discrete stable hexamers under con-ditions used to stabilize the therapeutic prepara-tion (i.e. in the presence of both phenolicpreservatives and zinc).

Interestingly, clinical studies have shown that thetime-action of LysB2 8ProB29-human insulinremains unaltered in spite of the formation of dis-crete hexamers. The solution of the T3R f crystalstructure (where T refers to an extended confor-mation of the first eight residues of the B-chain,and Rf describes an extended conformation forthe first three residues and an o-helical one forresidues four to eight), prepared in the presence ofzinc and phenol, has provided an explanation asto why the rapid time-action of LysB 28ProB2 9 -

human insulin in the hexameric state is unalteredin the therapeutically acceptable preparation. TheT3Rf crystal structure, although isomorphouswith that of T3R3f human insulin, clearly exhibitedlocalized structural differences in the C-terminalregion of the B-chain. These differences result inthe elimination of two critical hydrophobic inter-actions involving ProB2 8 and a weakening of two[3-pleated sheet hydrogen bonds that stabilize thedimer subunits that compose the hexamer. Theselocalized structural changes coupled with therequirement of phenol and zinc for the stabiliza-tion of the hexamer explain why the formulatedLysB28 ProB2 9-human insulin hexamer can readilydissociate in the subcutaneous tissue, yielding themonomeric form of the analog which is rapidlyabsorbed. We hypothesize that the absorption ofthe phenolic preservative destabilizes the hexamer,allowing it to dissociate into monomers at milli-molar concentrations.

Materials and methodsCrystallizationBiosynthetic LysB2 8ProB29 -human insulin was provided by theBiosynthetic Development Division of the Lilly Research Lab-oratories. Single crystals from which the 2.5 A data were mea-sured were grown from a solution containing 0.27 mMLysB28proB29-human insulin, 0.12 M acetic acid, 0.05 Msodium citrate in the presence of Zn2+ and phenol at a pH of5.9, by slow cooling from 500C to room temperature. As crys-tallization experiments were continued, larger single crystalsgrew under similar conditions, in which the 0.12 M aceticacid was replaced by 0.12 M hydrochloric acid; these crystalswere used to measure the 2.3 A data.

X-ray data collectionThe two sets of diffraction data were measured using anR-AXIS II-C image plate system and RU-200 rotating anodegenerator with CuKxo radiation. Integration, interframe scaling,and merging were performed with the R-AXIS software.

The 2.5 A data were measured from a single crystal of dimen-sions 0.20 mmX0.15mmX0.15mm and a total of 3306 mea-surements yielded 2653 unique data [Rmerge(F 2 )=0.069]. Thecrystals belong to space group R3 and were indexed in ahexagonal unit cell. The volume of the unit cell of dimensionsa=b=79.62 A, c=37.78 A is consistent with the presence of adimer in the asymmetric unit, and the crystals are nearly iso-morphous with crystals of uncomplexed T3R3f human insulin[3]. A second set of diffraction data was measured from a crystalof dimensions 0.20 mm x 0.30 mm x 0.30 mm, to a resolu-tion of 2.3 A. A total of 19299 data yielded 3910 unique data[Rmerge (F2)=0.060].

Structure solution and refinementThe coordinates of a T3R3f human insulin dimer [3], excludingresidues B1, B2 and B27-B30 of both B-chains, zinc, chlorideions, and water molecules, were used to calculate an initial dif-ference electron-density map using the 2.5 A data. The largestpeaks from this map corresponded to a pair of zinc ions and achloride ion located on the threefold axis. The next largestregion of electron density was found at the positions expectedfor residues ValB2 and ThrB 27. Initial refinement of the modelwas performed using the programs X-PLOR [16] andPROFFT [17,18]. When the 2.3 A data became available, therefinement was stopped at a residual of 0.181.

Table 3. Refinement statistics.

Model Target a

Distances (A)Bond 1-2 0.016 0.020Angle 1-3 0.054 0.050Planar 1-4 0.046 0.050

Chiral volume (A3) 0.175 0.150Planar groups (A) 0.013 0.020Thermal parameters (A2)

<AB> Main chain 2.156 2.500<AB> Side chain 1.555 1.500

Non-bonded distances (A)Single torsion 0.235 0.500Multiple torsion 0.268 0.500Possible H-bonds 0.412 0.500

Torsion angles (°)Planar 2.1 2.5Staggered 18.4 20.0Orthonormal 15.7 20.0

622 Structure 1995, Vol 3 No 6

GluB2t 2, ThrB27 2 and ThrB302. The refinement converged ata residual of 0.161 for 3548 data [Fo>2((F)] between 8.0 Aand 2.3 A resolution, and 0.178 for all 3860 data in this range.Details of the refinement are given in Table 3. Illustrated inFigure 8 is the 2Fo-F c electron density in the vicinity of the Ctermini of the B-chains of both monomers.

The coordinates have been deposited with the BrookhavenProtein Data Bank (entry code 1LPH).

Acknowledgements: The authors wish to thank Walter A Pangbornfor assistance in the data collection. EC was on leave from theUniversity of Opole, Opole, Poland. This work has been sup-ported in part by a Grant from Lilly Research Laboratories. Dedi-cated to the memory of Dorothy Hodgkin, whose insight madethis work possible.

Fig. 8. 2Fo-F electron density maps contoured at lo- in the vicin-ity of the B-chain C-terminal residues of (a) the T-state monomerand (b) the Rf-state monomer.

A structure factor calculation, using the second data set and therefined coordinates from the 2.5 A refinement, produced aresidual of 0.219. The program PROFFT [17,18] supported bythe graphics program CHAIN [19] was used to refine both thepositional and thermal parameters of the structure. The missingfragments (PheBI and LysB28-Thr 33 of both monomers) werelocated during the refinement based on the l (2Fo-F) elec-tron-density maps and were subsequently included into therefinement. Appropriate adjustments were made to main andside chains, and water molecules were added in accord withthe criteria of good electron density and acceptable hydrogenbonds to other atoms. No interpretable electron density wasobserved for the side-chain atoms of Tyr A I4.1, Thr B3 1l 1,

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Received: 13 Mar 1995; revisions requested: 30 Mar 1995;revisions received: 7 Apr 1995. Accepted: 7 Apr 1995.