Click here to load reader

Structural model of the nucleotide-binding ... Proc. Nati. Acad. Sci. USA Vol. 88, pp. 84-88, January 1991 Biochemistry Structural modelofthe nucleotide-binding conservedcomponentof

  • View
    1

  • Download
    0

Embed Size (px)

Text of Structural model of the nucleotide-binding ... Proc. Nati. Acad. Sci. USA Vol. 88, pp. 84-88,...

  • Proc. Nati. Acad. Sci. USA Vol. 88, pp. 84-88, January 1991 Biochemistry

    Structural model of the nucleotide-binding conserved component of periplasmic permeases

    (molecular modelng/sequence alinent/membrane transport/traffic ATPases)

    CAROL S. MIMURA*, STEPHEN R. HOLBROOKt, AND GIOVANNA FERRO-LUZZI AMES*t *Division of Biochemistry and Molecular Biology, and tChemical Biodynamics Division, University of California, Berkeley, CA 94720

    Communicated by Howard K. Schachman, September 26, 1990 (receivedfor review July 24, 1990)

    ABSTRACT The amino acid sequences of 17 bacterial membrane proteins that are components of periplasmic per- meases and function in the uptake of a variety of small molecules and ions are highly homologous to each other and contain sequence motifs characteristic of nucleotide-binding proteins. These proteins are known to bind ATP and are postulated to be the energy-coupling components of the per- meases. Several medically important eukaryotic proteins, in- cluding the multidrug-reslstance transporters and the protein encoded by the cystic fibrosis gene, are also homologous to this family. By multiple sequence alignment ofthese 17 proteins, the consensus sequence, secondary structure, and surface exposure were predicted. The secondary structural motifs that are conserved among nucleotide-binding proteins were identified in adenylate kinase, p21rw, and elongation factor Tu by superposition of their known tertiary structures. The equiva- lent secondary structural elements in the predicted conserved component were located. These, together with sequence infor- mation, served as guides for alignment with adenylate kinase. A model for the structure of the ATP-bindlng domain of the permease proteins is proposed by analogy to the adenylate kinase structure. The characteristics of several permease mu- tations and biochemical data lend support to the model.

    Periplasmic active transport systems (permeases) in Gram- negative bacteria transport a wide variety of substrates, including amino acids, peptides, ions, carbohydrates, and vitamins. These permeases share a common organization consisting of a substrate-binding protein that is located in the periplasm and imparts substrate specificity and a membrane- bound complex consisting of two hydrophobic membrane- spanning proteins plus a third membrane protein with a hydrophilic sequence (1). When different permeases are compared, the hydrophilic membrane protein (hereafter re- ferred to as the conserved component) invariably displays large stretches of sequence similarity, two of which are homologous to a previously defined ATP-binding consensus (1, 2), such as found in the a and f3 subunits of the FoF, ATPase, myosin, adenylate kinase, and others (3). This finding suggested that the function of the conserved compo- nent is to couple the energy of ATP hydrolysis to active transport. Indeed, recent experiments showed that ATP (and GTP) bind to the conserved components (4, 5) and that ATP hydrolysis is coupled to active transport (6-9). The con- served component has also been implicated in an interaction with the substrate-binding protein (10). Thus, each conserved component must include unique domains reflecting its inter- action with the individual hydrophobic membrane proteins and the specific periplasmic protein.

    Besides periplasmic permeases, other transport-related proteins are also homologous to the conserved components,

    indicating that these proteins constitute a superfamily with a common evolutionary origin and/or biochemical function. It has been proposed that members of this family be called "traffic ATPases" because they transport a variety of sub- strates at the expense of ATP and they translocate in both directions (11). Among these are several eukaryotic proteins such as the family of multidrug-resistance transporters of tumor cells (Mdr), the yeast protein STE6 responsible for secretion of the mating a-factor, the cystic fibrosis gene product (CFT-R), and others. To understand the molecular mechanism of action of the

    bacterial permeases and of the related eukaryotic proteins, it is necessary to know the structure of the conserved compo- nents. However, none ofthese proteins has been crystallized. Therefore, we have initiated a structure-function analysis by predicting the three-dimensional structure of the conserved components. Since proteins that share a common function are expected to conserve the three-dimensional folding pat- tern required for that function even though little sequence similarity may exist, we have determined the structural and functional constraints in common between the conserved components and proteins of known structure. A sequence alignment ofthe conserved components was performed, from which the consensus sequence, secondary structure, and surface exposure pattern were predicted. Then, based on a comparison of the primary and secondary structure with the known structures of the nucleotide-binding proteins adenyl- ate kinase, elongation factor Tu (EF-Tu), and p2115, a three-dimensional model was inferred for the conserved components and in particular for the histidine permease conserved component, HisP.

    METHODS Sequences were initially compared with the program GEN- ALIGN (12) constraining the nucleotide-binding motifs to align. Multiple sequence alignment utilized the method of Vingron and Argos (13). Secondary structure prediction for each pro- tein was made using both the Chou-Fasman approach (14) and a modification of the neural network method of Qian and Sejnowski (15). The surface accessibility was predicted by the method of Holbrook et al. (16). Computer graphics visualiza- tions ofprotein models as well as molecular superpositions and model building were done with the program INSIGHT.

    RESULTS AND DISCUSSION Sequence Aliment of the Conserved Components. The

    sequences of 17 conserved components (for review, see refs. 1 and 11; two of the protein, RbsA and AraG, have been divided into the amino and carboxyl halves because each half contains a nucleotide-binding consensus) were compared to one another through the repeated use of GENALIGN and were grouped in clusters as follows: (1) HisP and GlnQ; (2) AraG(C), RbsA(C), and CysA; (3) PstB, SfuC, UgpC, ProV,

    Abbreviation: EF, elongation factor. tTo whom reprint requests should be addressed.

    84

    The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

    D ow

    nl oa

    de d

    by g

    ue st

    o n

    F eb

    ru ar

    y 2,

    2 02

    1

  • Biochemistry: Mimura et al.

    FhuC, AraA(N), and RbsA(N); (4) ChID, OppD, and OppF; (5) MalK; (6) BtuD. After a prealignment of the sequences within clusters 1-4, the multiple sequence alignment shown in Fig. 1 was built up by consecutive additions of the subgroups to the growing list. The consensus sequence is shown at the bottom. Nine gaps in the consensus sequence are due to insertion of extra residues in individual proteins, presumably corresponding to segments related to special functions. Regions of similarity presumably reflect segments with common structure and function. Two regions of partic-

    Proc. Natl. Acad. Sci. USA 88 (1991) 85

    ularly high similarity (residues 52-63 and residues 183-221) clearly stand out and correspond to the well conserved nucleotide-binding motif (3). A particularly variable region occurs around residue 100 and will be discussed below.

    Prediction of Consensus Structural Features. The consensus prediction of secondary structure is illustrated schematically in Fig. 1. The conserved regions correspond to conserved structural motifs discussed below. A striking feature is the large helical domain consisting of four a-helices (H1 to H4) totaling 99 residues that are predicted to occur consecutively

    HisP t 10 20 30 40 50 60 70 80 90 100 110 120 HisP MMSENKLHVIDLHKRY------------- GGHEVLKGVSLQARAGDVISI IGSSGSGKSTFLRCINFL---P--EXPSEGAI IVNGQNINLVRDKDGQLKVADKNQLRLLRTRLTMVFQHF--NLWSHMTVLENV--MEAPIQ ----- VLG GlnQ -----MIEFKNVSKHF------------ GPTQVLHNIDLNIAQGEVVVI IGPSGSGKSTLLRCINKL-----EEITSGDLIV------------GDLKVNDPKVDERLIRQEAGMVFQQ--FYLFPHLTALEN--VMFGPL.-----RVR CysA ----MSIEIANIKKSF-------------GRTQVLNDISLDIPSGQMVALLGPSGSGKTTLLRI IAGL----EHQTSGHIRFHGTDVS---------------RLHARDRKVGFVFQHY--ALFRHMTVFDNIAFGLTVLP----- RRE RbsA APGDIRLKVDNLCGP -----------------GVND-VSFTLRKGEILGVSGLMGAGRTELMKVLYGA---- LPRTSGYVTLDGHEWTRSPQDG---LANGIVYISEDRXRDGLVLGM--SVKENMSLTALRYFSRAGGS-----LKH AraG< SYGEERLRLDAVKAP-----------------GVRTPISLAVRSGEIVGLFGLVGAGRSELMKGMFGG-----TQOITAGQVYIDQQPIDIRKPSHA--- IAAGMMLCPEDRKAEGI IPVH--SVRDNINISARRKHVLGGCV-----INN FhuC< NHSDTTFALRNISFRV------PGRTLLHPLSLTFPAGKVTGLIGHNGSGKSTLLKMLGRH---- QPPSEGEILLDAQPLE--------- SWSSKA----FARKVAYLPQQL--PPAEGMTVRELVAIGRYPWHGALGRFGA SfuC ---MSTLELHGIGKSY------------- NAIRVLEHIDLQVAAGSRTAIVGPSGSGKTTLLRIIAGF -----EIPDGGQILLQGQAMG-----------NGSGWVPAHLRGIGFVPQDG--ALFPHFTVAGNIGFGL----------- K ProV< EKGLSKEQILEKT----------------GLSLGVKDASLAIEEGEIFVIMGLSGSGKSTMVRLLNRL----- IEPTRGQVLIDGVDIA---------KISDAELREVRRKKIAMVFQSF--ALMPHMTVLDNTAFGM-----ELAGIAA UgpC ---MAGLKLQAVTKSW------------ DGKTQVIKPLTLDVADGEFIVMVGPSGCGKSTLLRMVAGL----ERVTEGDIWINDQRVT--------- EMEPKD------ RGIAMVFQNY--ALYPHMSVEENMAWGL-----KIRGMGK PstB< ETAPSKIQVRNLNFYY.-------.------GKFHALKNINLDIAXNQVTAFIGPSGCGKSTLLRTFNKMFELYPEQRAEGEILLDGDNI-----------LTNSQDIALLRAKVGMVFQKP--TPFP-MSIYDNIAFGV---- RLFKLSR RbsA --MEALLQLKGIDKAF-----------PG-VKALSGAALNVYPGRVMALVGENGAGKSTMMKVLTGI-----YTRDAGTLLWLGKETTFTGPKSSQEA- -----------GIGIIHQEL--NLIPQLTIAENIFLGR-EFVNRFGKIDW AraG< QQSTPYLSFRGIGKTF-----------PG-VKALTDISFDCYAGQVH