Functional Insights from the Structure of the Multifunctional C345C Domain of C5 of Complement
2004; Elsevier BV; Volume: 280; Issue: 11 Linguagem: Inglês
10.1074/jbc.m413126200
ISSN1083-351X
AutoresJanice Bramham, Chuong-Thu Thai, Dinesh C. Soares, Dušan Uhrı́n, Ronald T. Ogata, Paul N. Barlow,
Tópico(s)Hemoglobinopathies and Related Disorders
ResumoThe complement protein C5 initiates assembly of the membrane attack complex. This remarkable process results in lysis of target cells and is fundamental to mammalian defense against infection. The 150-amino acid residue domain at the C terminus of C5 (C5-C345C) is pivotal to C5 function. It interacts with enzymes that convert C5 to C5b, the first step in the assembly of the membrane attack complex; it also binds to the membrane attack complex components C6 and C7 with high affinity. Here a recombinant version of this C5-C345C domain is shown to adopt the oligosaccharide/oligonucleotide binding fold, with two helices packed against a five-stranded β-barrel. The structure is compared with those from the netrin-like module family that have a similar fold. Residues critical to the interaction with C5-convertase cluster on a mobile, hydrophobic inter-strand loop that protrudes from the open face of the β-barrel. The opposite, helix-dominated face of C5-C345C carries a pair of exposed hydrophobic side chains adjacent to a striking negatively charged patch, consistent with affinity for positively charged factor I modules in C6 and C7. Modeling of homologous domains from complement proteins C3 and C4, which do not participate in membrane attack complex assembly, suggests that this provisionally identified C6/C7-interacting face is indeed specific to C5. The complement protein C5 initiates assembly of the membrane attack complex. This remarkable process results in lysis of target cells and is fundamental to mammalian defense against infection. The 150-amino acid residue domain at the C terminus of C5 (C5-C345C) is pivotal to C5 function. It interacts with enzymes that convert C5 to C5b, the first step in the assembly of the membrane attack complex; it also binds to the membrane attack complex components C6 and C7 with high affinity. Here a recombinant version of this C5-C345C domain is shown to adopt the oligosaccharide/oligonucleotide binding fold, with two helices packed against a five-stranded β-barrel. The structure is compared with those from the netrin-like module family that have a similar fold. Residues critical to the interaction with C5-convertase cluster on a mobile, hydrophobic inter-strand loop that protrudes from the open face of the β-barrel. The opposite, helix-dominated face of C5-C345C carries a pair of exposed hydrophobic side chains adjacent to a striking negatively charged patch, consistent with affinity for positively charged factor I modules in C6 and C7. Modeling of homologous domains from complement proteins C3 and C4, which do not participate in membrane attack complex assembly, suggests that this provisionally identified C6/C7-interacting face is indeed specific to C5. A complement-mediated response to infection is fundamental to good health, but inappropriate complement activity underlies the symptoms of numerous inflammatory disorders (1Mizuno M. Morgan B.P. Curr. Drug Targets Inflamm. Allergy. 2004; 3: 87-96Crossref PubMed Scopus (29) Google Scholar). Activation of complement, and the ensuing attack on pathogens, entails a sequence of intermolecular recognition events, enzymatic cleavages, and assemblies of multiprotein complexes. The ∼30 fluid-phase and membrane-associated proteins participating in the complement system have been well characterized at the sequence level, and their respective roles are broadly understood (2Walport M.J. N. Engl. J. Med. 2001; 344: 1140-1144Crossref PubMed Scopus (1275) Google Scholar, 3Walport M.J. N. Engl. J. Med. 2001; 344: 1058-1066Crossref PubMed Scopus (2427) Google Scholar). There is, however, little understanding at atomic resolution of the interplay between the components. In particular, the sequence in which the five soluble, terminal components of complement (C5, C6, C7, C8, and C9) assemble to form the remarkable lipid bilayer-penetrating membrane attack complex (MAC) 1The abbreviations used are: MAC, membrane attack complex; C5-C345C, residues Ala1512 to the C-terminal Cys1658 of human C5; FIMAC, factor I membrane attack complex; OB, oligosaccharide/oligonucleotide binding; NOE, nuclear Overhauser effect; NOESY, NOE spectroscopy; NTR, netrin-like; PCOLCE, type I procollagen C-proteinase enhancer protein; r.m.s.d., root mean square deviation; SPR, surface plasmon resonance; TIMP, tissue inhibitor of metalloproteinase; WT, wild type; PDB, Protein Data Bank. has been known for many years (4Muller-Eberhard H.J. Biochem. Soc. Symp. 1985; 50: 235-246PubMed Google Scholar). But the network of protein-protein interactions entailed in forming this stable lytic complex and the involvement of specific amino acids remain a mystery. The key to progress in this area will be more three-dimensional structural information. Assembly of the MAC is initiated by proteolytic cleavage of C5 by the trimeric enzyme, C5 convertase, at the target cell surface to generate C5a and a metastable species, C5b. C5b has the transient ability to interact tightly with C6 (5DiScipio R.G. Smith C.A. Muller-Eberhard H.J. Hugli T.E. J. Biol. Chem. 1983; 258: 10629-10636Abstract Full Text PDF PubMed Google Scholar). The C5bC6 complex subsequently serves as a nucleation site for sequential assembly of C7, C8, and n molecules of C9 to create the MAC. Mature C5 is a heterodimer consisting of α and β chains of 115 and 75 kDa, respectively. It is evolutionarily related to the earlier complement components C3 and C4. Unlike the majority of proteins of the complement system, C3, C4, and C5 are not entirely modular in their composition. Nonetheless it is surprising, given the intense interest in this family of proteins that has persisted over several decades, that little is known of their structure. For example, although the solution structure of the much smaller C5a fragment has been solved (6Zuiderweg E.R. Nettesheim D.G. Mollison K.W. Carter G.W. Biochemistry. 1989; 28: 172-185Crossref PubMed Scopus (97) Google Scholar), in the case of C5b there is currently no three-dimensional structural information available. An opportunity to address this lack of structural information arose from the suggestion that the C-terminal ∼150-amino acid residue portions of the α chains of C3 and C5 and of the γ chain of C4 are independently folded units (7Ishii N. Wadsworth W.G. Stern B.D. Culotti J.G. Hedgecock E.M. Neuron. 1992; 9: 873-881Abstract Full Text PDF PubMed Scopus (434) Google Scholar, 8Banyai L. Patthy L. Protein Sci. 1999; 8: 1636-1642Crossref PubMed Scopus (149) Google Scholar). Not only do these regions exhibit high sequence similarity with one another but they also display an ∼19% sequence identity to the C-terminal segment of the Caenorhabditis elegans UNC-6, a laminin-related netrin protein involved in axonal path finding (7Ishii N. Wadsworth W.G. Stern B.D. Culotti J.G. Hedgecock E.M. Neuron. 1992; 9: 873-881Abstract Full Text PDF PubMed Scopus (434) Google Scholar). Furthermore, homologies with C-terminal segments of procollagen C-proteinase enhancer proteins (PCOLCEs) and of secreted frizzled-related proteins have been noted (8Banyai L. Patthy L. Protein Sci. 1999; 8: 1636-1642Crossref PubMed Scopus (149) Google Scholar), and this family of domains has been named the NTR (netrin-like) module. The more recently solved three-dimensional structure of the PCOLCE-1 NTR module (9Liepinsh E. Banyai L. Pintacuda G. Trexler M. Patthy L. Otting G. J. Biol. Chem. 2003; 278: 25982-25989Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar) confirmed structural similarities with the N-terminal domains of tissue inhibitor of metalloproteinases (TIMP)-1 and -2 (10Muskett F.W. Frenkiel T.A. Feeney J. Freedman R.B. Carr M.D. Williamson R.A. J. Biol. Chem. 1998; 273: 21736-21743Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 11Fernandez-Catalan C. Bode W. Huber R. Turk D. Calvete J.J. Lichte A. Tschesche H. Maskos K. EMBO J. 1998; 17: 5238-5248Crossref PubMed Scopus (315) Google Scholar), the laminin-binding domain of agrin (12Stetefeld J. Jenny M. Schulthess T. Landwehr R. Schumacher B. Frank S. Ruegg M.A. Engel J. Kammerer R.A. Nat. Struct. Biol. 2001; 8: 705-709Crossref PubMed Scopus (40) Google Scholar), and the oligosaccharide/oligonucleotide-binding (OB) domains of some single-stranded DNA-binding proteins (13Murzin A.G. EMBO J. 1993; 12: 861-867Crossref PubMed Scopus (779) Google Scholar). Expression of the segment of C5 corresponding to its C345C domain (14Thai C.T. Ogata R.T. J. Immunol. 2003; 171: 6565-6573Crossref PubMed Scopus (23) Google Scholar) followed by analysis using CD and NMR confirmed that these amino acid residues fold to form a compact three-dimensional structure (15Bramham J. Rance M. Thai C.T. Uhrin D. Assa-Munt N. Ogata R.T. Barlow P.N. J. Biomol. NMR. 2004; 29: 217-218Crossref PubMed Scopus (1) Google Scholar). Furthermore, C5-C345C, unlike the C345C domain of C3, is able to bind to both C6 and C7 in surface plasmon resonance (SPR)-based assays (14Thai C.T. Ogata R.T. J. Immunol. 2003; 171: 6565-6573Crossref PubMed Scopus (23) Google Scholar). In further work, C5-C345C was shown to inhibit recruitment of C7 by C5bC6 through an interaction between C5-C345C and the pair of factor I membrane attack complex (FIMAC) domains, also called factor I modules (FIMs), at the C terminus of C7 (16Thai C.T. Ogata R.T. J. Immunol. 2004; 173: 4547-4552Crossref PubMed Scopus (39) Google Scholar). Thus the C5-C345C domain provides at least part of the interacting surface between C5b and C7 in formation of the MAC. The C345C domain also harbors a region that interacts with the C5 convertase (17Sandoval A. Ai R. Ostresh J.M. Ogata R.T. J. Immunol. 2000; 165: 1066-1073Crossref PubMed Scopus (31) Google Scholar), although the cleavage site itself lies some 800 residues away toward the N terminus of the α chain of C5. Although the fold of C5-C345C might be anticipated to resemble the fold of the NTR module from PCOLCE-1 (9Liepinsh E. Banyai L. Pintacuda G. Trexler M. Patthy L. Otting G. J. Biol. Chem. 2003; 278: 25982-25989Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar), the sequence identity is low (see Fig. 1A) and disulfide bonding patterns are different, 1–4, 2–5, and 3–6 in the PCOLCE-1 NTR module compared with 1–3, 2–6, and 4–5 in the C3 equivalent (and therefore by inference in the C5 example). The C5-C345C sequence is longer and contains fewer prolines (147 residues including three prolines) compared with the PCOLCE-1 NTR module sequence (119 residues including 11 prolines). An experimentally determined three-dimensional structure of C5-C345C would therefore represent an important advance in understanding the basis, at atomic resolution, for the early steps of MAC assembly. Here we report the use of solution NMR to solve the structure of C5-C345C. We thus provide the first new structural information for the C3/C4/C5 family of proteins since the structures of the C3d and C4d fragments were solved (18Nagar B. Jones R.G. Diefenbach R.J. Isenman D.E. Rini J.M. Science. 1998; 280: 1277-1281Crossref PubMed Scopus (163) Google Scholar, 19van den Elsen J.M. Martin A. Wong V. Clemenza L. Rose D.R. Isenman D.E. J. Mol. Biol. 2002; 322: 1103-1115Crossref PubMed Scopus (47) Google Scholar) and, in the case of C5, since the anaphylatoxic C5a fragment structure was determined in 1989 (6Zuiderweg E.R. Nettesheim D.G. Mollison K.W. Carter G.W. Biochemistry. 1989; 28: 172-185Crossref PubMed Scopus (97) Google Scholar). The new structure allows the construction of useful models of the C345C domains from C3 and C4. The positions within the structure of residues previously identified as being functionally critical and the location of surface patches likely to be involved in protein-protein interactions are now revealed. Protein Preparation—pET15b vectors encoding the amino acid residues of C5 from Ala1512 to the C-terminal residue Cys1658 (both with and without the point mutation F1613A) were constructed as described previously (14Thai C.T. Ogata R.T. J. Immunol. 2003; 171: 6565-6573Crossref PubMed Scopus (23) Google Scholar). The isotopically enriched recombinant proteins were overexpressed in the Escherichia coli strain Origami (Novagen, Madison, WI) and purified as described previously (14Thai C.T. Ogata R.T. J. Immunol. 2003; 171: 6565-6573Crossref PubMed Scopus (23) Google Scholar). For NMR studies, 15N- and 15N, 13C-protein samples (0.5–1.0 mm) were prepared in buffer containing 20 mm sodium phosphate, 100 mm NaCl, 5 μm EDTA, 0.02% NaN3, pH 6.0, in 95% H2O, 5% D2O. Binding Studies—Affinities of the recombinant wild-type and F1613A versions of C5-C345C for C6 and C7 were measured using SPR as described previously (14Thai C.T. Ogata R.T. J. Immunol. 2003; 171: 6565-6573Crossref PubMed Scopus (23) Google Scholar). NMR Spectroscopy—NMR spectra were acquired on Bruker AVANCE 600- and 800-MHz and Varian INOVA 600- and 800-MHz spectrometers, using 5-mm triple resonance probes equipped with pulse-field gradients. Spectra were processed using the AZARA package (provided by W. Boucher, University of Cambridge), using maximum entropy processing of F1 and F2 dimensions of the three-dimensional experiments, and resonance assignment was achieved using ANSIG as described previously (15Bramham J. Rance M. Thai C.T. Uhrin D. Assa-Munt N. Ogata R.T. Barlow P.N. J. Biomol. NMR. 2004; 29: 217-218Crossref PubMed Scopus (1) Google Scholar). Distance restraints for the structure calculation were derived from the following three complementary NOE spectroscopy (NOESY) experiments: a 15N-edited NOESY-HSQC and two 13C-edited NOESY-HSQCs, one in H2O buffer and one in D2O buffer. All mixing times were 100 ms. Slowly exchanging amide protons were identified by the detection of 26 NH resonances in a 15N-HSQC spectrum recorded 1 month after exchanging a protein sample into D2O buffer. Hydrogen bond acceptors for most of these slowly exchanging protons were identified using the refined initial structures. Distance restraints corresponding to hydrogen bonds were only introduced following identification of the supporting characteristic NOEs. 15N Relaxation Measurements—15N T1 and T2 relaxation times were measured by the method of Kay (20Kay L.E. Nicholson L.A. Delaglio F. Bax A. Torchia D.E. J. Magn. Reson. 1992; 97: 359-375Google Scholar). The pulse sequence was modified according to Grzesiek and Bax (21Grzesiek S. Bax A. J. Am. Chem. Soc. 1993; 115: 12593-12594Crossref Scopus (1015) Google Scholar) to keep the water magnetization on the z axis during the T1 period. Relaxation delays of 43.1, 253.1, 421.1, 589.1, 757.1, 841.1, 925.1, and 1051.1 ms were employed for T1 measurements, and delays of 15.8, 31.6, 63.2, 94.8, 111.7, and 126.5 ms were employed for T2 measurements. The T1 and T2 relaxation times were calculated by nonlinear least squares fitting. In each case, the spectrum corresponding to one of the relaxation delay values was re-collected to allow an estimation of the experimental error of the measured peak intensities. For the 1H-15N HSQC heteronuclear NOE experiment (21Grzesiek S. Bax A. J. Am. Chem. Soc. 1993; 115: 12593-12594Crossref Scopus (1015) Google Scholar), a saturation experiment and a reference experiment were recorded with a relaxation delay of 5 s, of which 3 s was used for 1H saturation in the 1H-saturated experiment. Structure Calculation—Wherever possible, resonances in the NOESY spectra were assigned unambiguously. Otherwise a set of two or more assignment possibilities were assigned on the basis of their chemical shifts using the Connect program within AZARA. Peak intensities were converted into four distance categories of 0–2.7, 0–3.3, 0–5.0, and 0–6.0 Å. A total of 3544 distance restraints were generated from the three NOESY-HSQCs, of which 2609 were unambiguous and nondegenerate. The NOE-derived distance restraints were used as input for the structure calculations using CNS-Solve (22Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16979) Google Scholar). At a later stage more distance restraints representing the 26 inferred hydrogen bonds were added to the restraints list. The simulated annealing protocol employed the PARALLHD force field, with the nonbonded energy function of PROLSQ (23Linge J.P. Nilges M. J. Biomol. NMR. 1999; 13: 51-59Crossref PubMed Scopus (239) Google Scholar) and included active swapping of pro-chiral centers. For the initial structure calculations, the six cysteines were defined as being in the oxidized state. In the absence of information from experimental disulfide mapping, however, no covalent linkages between sulfur atoms were initially defined in the molecular structure file in order to avoid bias. At a later stage, and based on the initial structure calculations, two disulfide bonds, Cys1514–Cys1588 and Cys1535–Cys1658, were added. There was a lack of NOE-based evidence to support the formation of a disulfide between the remaining pair of cysteines, Cys1636 and Cys1639. This arose, at least in part, from a paucity of assignments for nuclei in this region of the sequence. No covalent linkage was therefore defined between these residues. As the calculations progressed, the ambiguously assigned distance restraints were “filtered” iteratively to eliminate assignment possibilities contributing less than 1% to the total NOE, and redundant restraints (duplicates) were also removed. A total of 100 structures were calculated from which a representative ensemble of 40 structures, with the lowest NOE-derived energies, was selected. The quality of the ensemble of structures was checked with PROCHECK (24Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). The NOE-derived distance restraints used for the structure calculations and the coordinates of the ensemble of 40 structures of C5-C345C have been deposited in the Protein Data Bank under accession number 1XWE. Modeling C345C Domains of C3 and C4 —Modeling of the C345C domains of C3 and C4 was undertaken based on the lowest NOE energy NMR-derived structure of C5-C345C using the program Modeller release 7, version 7 (25Sali A. Blundell T.L. J. Mol. Biol. 1993; 234: 779-815Crossref PubMed Scopus (10636) Google Scholar). The alignments between the target sequences of human C3 and C4 C345C domains and the template structure were based on initial multiple sequence alignments of C3-, C4-, and C5-C345C domain sequences from various organisms from the SwissProt (26Boeckmann B. Bairoch A. Apweiler R. Blatter M.C. Estreicher A. Gasteiger E. Martin M.J. Michoud K. O'Donovan C. Phan I. Pilbout S. Schneider M. Nucleic Acids Res. 2003; 31: 365-370Crossref PubMed Scopus (2822) Google Scholar, 27O'Donovan C. Martin M.J. Gattiker A. Gasteiger E. Bairoch A. Apweiler R. Brief. Bioinform. 2002; 3: 275-284Crossref PubMed Scopus (221) Google Scholar) and the GenBank™ nonredundant databases, using the program MUSCLE (28Edgar R.C. BMC Central Bioinformatics. 2004; 5: 113Crossref PubMed Scopus (6100) Google Scholar, 29Edgar R.C. Nucleic Acids Res. 2004; 32: 1792-1797Crossref PubMed Scopus (31027) Google Scholar). The multiple sequence alignment (Fig. 1B) was manually edited to ensure the most plausible alignment of conserved amino acid residues and of secondary structure elements as predicted by PsiPred (30McGuffin L.J. Bryson K. Jones D.T. Bioinformatics. 2000; 16: 404-405Crossref PubMed Scopus (2777) Google Scholar) between the target and template. The three putative disulfide bridges and the longer predicted C-terminal α-helix of both the C3 and C4 C345C domains were restrained during model building. Twenty models were generated in each case, and the one with the lowest objective function score (25Sali A. Blundell T.L. J. Mol. Biol. 1993; 234: 779-815Crossref PubMed Scopus (10636) Google Scholar) was selected as the representative model. The representative model structures were protonated using the program REDUCE (31Word J.M. Lovell S.C. Richardson J.S. Richardson D.C. J. Mol. Biol. 1999; 285: 1735-1747Crossref PubMed Scopus (1110) Google Scholar) and were checked for valid stereochemistry using PROCHECK (24Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). Recombinant F1613A Mutant Binds C6/C7—The protein fragment, C5-C345C (residues Ala1512 to the C-terminal Cys1658 of human C5), with an N-terminal His tag was overexpressed in the E. coli strain Origami. The use of a bacterial expression system facilitated isotopic enrichment, and the Origami strain was selected because its oxidizing intracellular environment is conducive to formation of disulfide bonds (32Bessette P.H. Aslund F. Beckwith J. Georgiou G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13703-13708Crossref PubMed Scopus (526) Google Scholar). After thrombin cleavage of the His tag, four extra residues (Gly-Ser-His-Met) remained at the N terminus of the C5-C345C sequence. Protein expression levels in rich media were typically 4 mg liter-1 but only 0.5 mg liter-1 in Martek 9-labeled media. Yields were improved 4–5-fold using a construct with the point mutation F1613A. To assess any structural perturbations that might be introduced by such a mutation, 15N, 1H-HSQC spectra of 15N-labeled wild-type and F1613A C5-C345C samples were compared (data not shown). Nearly all the resonances coincide. Significant chemical shift differences were noted only for those peaks corresponding to residues located close in sequence to the mutation, namely Ile1609–Tyr1617; of these only Asn1612, Phe/Ala1613, and Ser1614 show major differences. This observation demonstrates the F1613A mutant of C5-C345C has a near identical structure to that of the native domain. To assay for functional activity, binding to C6 and C7 was measured (Fig. 2). As may be judged from the SPR-derived binding parameters (Table I), the affinities of the F1316A mutant for both these MAC components are similar or identical to those of the wild-type domain. Given its higher expression levels, the mutant was therefore used in the subsequent structural studies.Table IBinding parameters for wild-type and F1613A C5-C345C Values are derived from the data in Fig. 2.C6C7KDkonkoffKDkonkoffnmnmWT C5-C345C102 × 1042 × 10-433 × 1049 × 10-5F1613A C5-C345C92 × 1042 × 10-423 × 1046 × 10-5 Open table in a new tab The Solution Structure of C5-C345C Is Solved—The 15N and 15N, 13C-labeled samples of C5-C345C yielded high quality NMR spectra thus permitting the assignment of nearly all of the 15N, 13C, and 1H nuclei (15Bramham J. Rance M. Thai C.T. Uhrin D. Assa-Munt N. Ogata R.T. Barlow P.N. J. Biomol. NMR. 2004; 29: 217-218Crossref PubMed Scopus (1) Google Scholar). Only a few assignments were made for Ser1637, Ser1638, and the four non-native residues at the N terminus because they all gave rise to few detectable resonances. Several assignments for aromatic side chain atoms were missing, mainly due to overlapping signals; these were Tyr1543 (Cϵ and Hϵ), Tyr1611 (Cϵ and Hϵ), Phe1556 (Cζ and Hζ), Phe1615 (Cϵ, Hϵ, Cζ, and Hζ), Phe1642 (Cϵ and Hϵ), and Phe1654 (Cϵ, Cζ, and Hζ). Tyr1541 is unusual in that its Hδ and its Hϵ nuclei have nondegenerate chemical shifts; a strong chemical exchange peak between the resonances of Hδ1 and Hδ2 and between Hϵ1 and Hϵ2, indicates restricted rotation of its aromatic side chain (subsequently, the structure reveals that this side chain is indeed well buried within the core of the protein). The Hα atom of Leu1521 exhibits an unusually low chemical shift of 1.58 ppm (cf. average shift is 4.32 ppm). All three proline residues are in the trans conformation as evidenced by the differences in the chemical shifts, δCβ–δCγ of 4.03, 5.00, and 4.88 ppm for Pro1537, Pro1620, and Pro1631, respectively (δCβ–δCγ is 4.51 ± 1.17 ppm for trans and 9.64 ± 1.27 ppm for cis (33Schubert M. Labudde D. Oschkinat H. Schmieder P. J. Biomol. NMR. 2002; 24: 149-154Crossref PubMed Scopus (277) Google Scholar)), as well as strong NOE cross-peaks between the Hδs of the prolines and the Hα of the preceding residues. Subsequently, a structure calculation was undertaken using a total of 3544 NMR-derived distance restraints as detailed in Table II. Two disulfide (Cys1514–Cys1588 and Cys1535–Cys1658) bonds were added only after NOE-based calculations had established beyond a doubt the proximity and orientation of the contributing cysteine side chains. A third potential disulfide was not invoked because, although the remaining two cysteine residues are close in space, there is insufficient NOE-derived evidence to judge whether their side chains are appropriately juxtaposed. Similarly, distance restraints based on 26 inferred inter-β-strand H-bonds were not added until the later stages of the structure calculation.Table IIStructural statistics for the 40 lowest energy structuresEnsembleClosest to meanNo. of experimental restraints used (all)35443544NOE-derivedIntraresidue12751275Sequential531531Medium range (|i - j| ≤ 4)221221Long range (|i - j| > 4)582582Ambiguous909909Hydrogen bonds2626Coordinate r.m.s.d. (Å)All residuesaResidues 1512-1658Backbone heavy atoms1.1860.986All heavy atoms1.6781.419Excluding loopsbExcluding residues 1512, 1610-1615, and 1635-1639Backbone heavy atoms0.7840.576All heavy atoms1.2770.964Ramachandran assessment (%)Most favored region68.873.8Additionally allowed region27.024.8Generously allowed region3.51.4Disallowed region0.70a Residues 1512-1658b Excluding residues 1512, 1610-1615, and 1635-1639 Open table in a new tab A total of 40 structures, selected on the basis of lowest NOE-derived energy from 100 calculated ones, converged well in most regions as may be judged from a backbone overlay (Fig. 3A) and the values for r.m.s.d. in Table II. The r.m.s.d. of the Cα coordinates of the 40 selected structures from those of the mean structure are plotted in Fig. 4A as a function of residue number and compared with the distribution of 1H-1H NOEs (Fig. 4B). Significantly fewer than average NOEs are exhibited by two stretches of residues within the sequence (Ile1609–Phe1615 and Thr1635–Cys1639) and by the N-terminal residues Ala1512 and Asp1513. This is reflected in the elevated r.m.s.d. values of their Cαs and is also evident from inspection of the overlay in Fig. 3A. In the case of Ser1637 and Ser1638, the aforementioned lack of detectable amide signals would account in part for the dearth of NOEs.Fig. 4Convergence, number of distance restraints, and relaxation measurements.A, the r.m.s.d. of each residue (Cα) is plotted based on a superposition that excluded residues 1512, 1610–1615, and 1635–1639. The secondary structure is summarized by the schematic, where stripes indicate helices and solid shading indicates β-strands. B, the number of experimentally derived unambiguous distance restraints per residue. Shading scheme: black, sequential; gray, medium range |i - j| ≤4; white = long range |i - j| >4. C, 15N T1/T2versus residue number, for backbone amides. D, heteronuclear (15N, 1H) NOEs as a function of residue number. The secondary structure is summarized as in A.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Description of the Structure—For the purposes of the description below, and unless stated otherwise, a residue is designated as belonging to an α-helix or β-strand in C5-C345C if it is so defined in the majority of the 40 members of the ensemble according to the Kabsch and Sander (34Kabsch W. Sander C. Biopolymers. 1983; 22: 2577-2637Crossref PubMed Scopus (12421) Google Scholar) criteria, as implemented in MolMol (35Koradi R. Billeter M. Wuthrich K. J. Mol. Graphics. 1996; 14: 51-55Crossref PubMed Scopus (6498) Google Scholar). Two views of the fold of the closest-to-the-mean C5-C345C structure are shown in Fig. 3B. The core of the structure is an OB-class fold that is most easily thought of as two orthogonal three-stranded, antiparallel twisted β-sheets composed from strands AC-B-C and strands AN-D-E, where the superscripts N and C denote the N- and the C-terminal halves of strand A (Tyr1541–Val1552). There are two adjacent helices as follows: a short one (helix-1, Arg1530–Ala1534) composed of residues from near the N terminus of the module, and a longer and irregular one (helix-2, Leu1643–Leu1655) close to the C terminus of the module (and of the full-length protein). The two helices are tilted with respect to one another but are essentially aligned with, and lie against, the convex face of the AN-D-E sheet. Strand B (Val1557–Lys1568) extends beyond the AC-B-C sheet so that its C-terminal part participates in a four-stranded anti-parallel sheet BC-AN-D-E. In a small proportion of calculated structures, there are two segments to strand E, E1 (Ile1618–Pro1620) and E2 (Trp1626–Tyr1629), interrupted by coil. Strand E1, which is assigned (within MolMol) in only a few structures, forms a small parallel β-sheet with strand C (Glu1579–Lys1584). In all of the C5-C345C structures, there is potential for H-bonds between the CO of Tyr1619 and the NH of Thr1581, and between the NH of residue Tyr1619 and CO of Ile1583, thus completing the hydrogen bond network that forms the barrel-like structure. Strand E2, which appears in all structures, is antiparallel to strand D (Gln1598–Gly1603). Thus the barrel has a “closed” side made up from the β-strands, and a more “open” side (to the right of the view in the left-hand panel of Fig. 3B) occupied by Tyr1619 and the residues prior to E2. The N-terminal s
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