A ‘Collagen Hug’ Model for Staphylococcus aureus CNA binding to collagen
2005; Springer Nature; Volume: 24; Issue: 24 Linguagem: Inglês
10.1038/sj.emboj.7600888
ISSN1460-2075
AutoresYinong Zong, Yi Xu, Xiaowen Liang, Douglas R. Keene, Agneta Höök, Shivasankarappa Gurusiddappa, Magnus Höök, Sthanam V.L. Narayana,
Tópico(s)Monoclonal and Polyclonal Antibodies Research
ResumoArticle15 December 2005free access A ‘Collagen Hug’ Model for Staphylococcus aureus CNA binding to collagen Yinong Zong Yinong Zong School of Optometry and Center for Biophysical Sciences and Engineering, University of Alabama at Birmingham, Birmingham, AL, USA Search for more papers by this author Yi Xu Yi Xu Center for Extracellular Matrix Biology, Institute of Biosciences and Technology, Texas A&M University Health Science Center, Houston, TX, USA Search for more papers by this author Xiaowen Liang Xiaowen Liang Center for Extracellular Matrix Biology, Institute of Biosciences and Technology, Texas A&M University Health Science Center, Houston, TX, USA Search for more papers by this author Douglas R Keene Douglas R Keene Shriners Hospital for Children, Portland, OR, USA Search for more papers by this author Agneta Höök Agneta Höök Center for Extracellular Matrix Biology, Institute of Biosciences and Technology, Texas A&M University Health Science Center, Houston, TX, USA Search for more papers by this author Shivasankarappa Gurusiddappa Shivasankarappa Gurusiddappa Center for Extracellular Matrix Biology, Institute of Biosciences and Technology, Texas A&M University Health Science Center, Houston, TX, USA Search for more papers by this author Magnus Höök Corresponding Author Magnus Höök Center for Extracellular Matrix Biology, Institute of Biosciences and Technology, Texas A&M University Health Science Center, Houston, TX, USA Search for more papers by this author Sthanam V L Narayana Corresponding Author Sthanam V L Narayana School of Optometry and Center for Biophysical Sciences and Engineering, University of Alabama at Birmingham, Birmingham, AL, USA Search for more papers by this author Yinong Zong Yinong Zong School of Optometry and Center for Biophysical Sciences and Engineering, University of Alabama at Birmingham, Birmingham, AL, USA Search for more papers by this author Yi Xu Yi Xu Center for Extracellular Matrix Biology, Institute of Biosciences and Technology, Texas A&M University Health Science Center, Houston, TX, USA Search for more papers by this author Xiaowen Liang Xiaowen Liang Center for Extracellular Matrix Biology, Institute of Biosciences and Technology, Texas A&M University Health Science Center, Houston, TX, USA Search for more papers by this author Douglas R Keene Douglas R Keene Shriners Hospital for Children, Portland, OR, USA Search for more papers by this author Agneta Höök Agneta Höök Center for Extracellular Matrix Biology, Institute of Biosciences and Technology, Texas A&M University Health Science Center, Houston, TX, USA Search for more papers by this author Shivasankarappa Gurusiddappa Shivasankarappa Gurusiddappa Center for Extracellular Matrix Biology, Institute of Biosciences and Technology, Texas A&M University Health Science Center, Houston, TX, USA Search for more papers by this author Magnus Höök Corresponding Author Magnus Höök Center for Extracellular Matrix Biology, Institute of Biosciences and Technology, Texas A&M University Health Science Center, Houston, TX, USA Search for more papers by this author Sthanam V L Narayana Corresponding Author Sthanam V L Narayana School of Optometry and Center for Biophysical Sciences and Engineering, University of Alabama at Birmingham, Birmingham, AL, USA Search for more papers by this author Author Information Yinong Zong1,‡, Yi Xu2,‡, Xiaowen Liang2, Douglas R Keene3, Agneta Höök2, Shivasankarappa Gurusiddappa2, Magnus Höök 2 and Sthanam V L Narayana 1 1School of Optometry and Center for Biophysical Sciences and Engineering, University of Alabama at Birmingham, Birmingham, AL, USA 2Center for Extracellular Matrix Biology, Institute of Biosciences and Technology, Texas A&M University Health Science Center, Houston, TX, USA 3Shriners Hospital for Children, Portland, OR, USA ‡These authors contributed equally to this work *Corresponding authors: Center for Biophysical Sciences and Engineering, School of Optometry, University of Alabama at Birmingham, 1025 18th Street South, Birmingham, AL 35294, USA. Tel.: +1 205 934 0119; Fax: +1 205 975 0538; E-mail: [email protected] for Extracellular Matrix Biology, Institute of Biosciences and Technology, Texas A&M University Health Science Center, 2121 W. Holcombe Blvd, Houston, TX 77030-3303, USA. Tel.: +1 713 677 7552; Fax: +1 713 677 7576; E-mail: [email protected] The EMBO Journal (2005)24:4224-4236https://doi.org/10.1038/sj.emboj.7600888 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The structural basis for the association of eukaryotic and prokaryotic protein receptors and their triple-helical collagen ligand remains poorly understood. Here, we present the crystal structures of a high affinity subsegment of the Staphylococcus aureus collagen-binding CNA as an apo-protein and in complex with a synthetic collagen-like triple helical peptide. The apo-protein structure is composed of two subdomains (N1 and N2), each adopting a variant IgG-fold, and a long linker that connects N1 and N2. The structure is stabilized by hydrophobic inter-domain interactions and by the N2 C-terminal extension that complements a β-sheet on N1. In the ligand complex, the collagen-like peptide penetrates through a spherical hole formed by the two subdomains and the N1–N2 linker. Based on these two structures we propose a dynamic, multistep binding model, called the ‘Collagen Hug’ that is uniquely designed to allow multidomain collagen binding proteins to bind their extended rope-like ligand. Introduction The collagens are the most abundant proteins in vertebrates. This class of proteins provides the structural support for tissues, serves as a scaffold for the assembly of extracellular matrices (ECM), and can also directly affect cell behavior through specific cellular receptors. Over 20 genetically different collagen types have been identified, in addition to a number of proteins that contain collagenous subdomains. The collagenous domains have a characteristic triple helix structure where each of the participating polypeptides are composed of repeating Gly-X-Y sequences that form hetero or homo-trimeric L-proline helices. The limited amino-acid sequence variations and the rope-like triple-helical structure of collagens present a unique challenge to the elucidation of the molecular and structural details of protein–collagen interactions. Currently, a plethora of proteins are known to interact with collagens; however, the mechanisms by which these interactions occur remain poorly understood. Both eukaryotes and prokaryotes express collagen-binding proteins, which include noncollagenous components of the ECM, cellular receptors such as some integrins, and bacterial collagen adhesins. Collagen degrading enzymes such as the eukaryotic matrix metalloproteinases (MMP) and the bacterial collagenases also contain collagen-binding domains. So far the I domain of integrin α2 (α2I) is the only structure of a collagen binding protein in complex with a collagen-derived peptide ligand that has been solved (Emsley et al, 2000). In this structure, an Mg2+ ion facilitates the protein–protein interaction by coordinating residues in the metal ion-dependent adhesion site (MIDAS) of α2I and a Glu residue in the collagen peptide. A family of structurally related collagen binding adhesins of the MSCRAMM (microbial surface component recognizing adhesive matrix molecules) type is found on Gram-positive pathogens such as Staphylococcus aureus (Patti et al, 1993), Enterococcus faecalis (Rich et al, 1999c), Enterococcus faecium (Nallapareddy et al, 2003), Streptococcus equi (Lannergard et al, 2003), Streptococcus mutans (Sato et al, 2004), Erysipelothrix rhusiopathiae (Shimoji et al, 2003), and Bacillus anthracis (Xu et al, 2004). These adhesive proteins from Gram-positive bacteria do not share sequence homology with the collagen binding I domains of integrins and do not require metal ions for collagen binding. Thus, these proteins appear to employ a collagen binding mechanism that maybe drastically different from that of the collagen binding integrins. The collagen binding MSCRAMM on S. aureus is called CNA and is the prototype member of this family. CNA participates in the infectious process of pathogenic S. aureus and is shown to be a virulence factor in many different animal models of staphylococcal infections including arthritis, endocarditis, osteomyelitis, mastitis and keratitis (Patti et al, 1993, 1994; Hienz et al, 1996; Mamo et al, 2000; Rhem et al, 2000; Elasri et al, 2002), suggesting that the ability to interact with collagen provides a general advantage to the bacteria in pathogenesis. Furthermore, the recombinant CNA can be used as an effective vaccine component and antibodies raised against CNA are protective in a mouse model of S. aureus induced septic death (Nilsson et al, 1998). CNA is composed of a so-called A-region and a varying number of B-repeats depending on the strain (Figure 1A). At the C-terminal end of CNA are features required for surface targeting and covalently anchoring to the peptidoglycan. The collagen binding activity is located in the A-region (Patti et al, 1993). Earlier studies from our laboratories have shown that a central segment of the A region contains the minimal collagen binding site and its crystal structure revealed a single domain representing a novel variant of the IgG-fold. Molecular modeling and docking experiments helped us to identify a grove on the surface of this domain as a putative collagen-binding site (Symersky et al, 1997). However, this central segment has a 10-fold lower affinity for collagen compared to the full length A-region, suggesting that regions flanking the central segment significantly participate in the collagen interaction (Patti et al, 1993; Xu et al, 2004). Figure 1.(A) The domain organization of S. aureus CNA and different constructs. The collagen binding A region is followed by B repeats. S, signal peptide; W, cell wall anchoring region containing the LPETG motif; M, transmembrane segment; and C, cytoplasmic tail. The three subdomains of A-region are from residues 31–140 (N1), 141–344 (N2), and 345–531 (N3). The previously identified minimum collagen-binding domain is from residues 151–318. CNA fragments constructed as N-terminal His-tag fusion proteins are illustrated. (B) Representative Biacore sensorgrams of different CNA fragments passed over collagen. The same concentration of purified CNA fragments (10 μM) was passed over a bovine type I collagen-coated surface. Injection started at ∼140 s and ended at ∼550 s. Responses from a blank surface were subtracted from the responses from the collagen-coated surface. (C) Inhibition of the binding of CNA31–344 to type I collagen by different CNA fragments. Biotin-labeled CNA31–344 (100 nM) was mixed with increasing concentrations of unlabeled CNA31-344 (inverted triangles), CNA31–531 (squares), and CNA151–318 (open circles), and then incubated in wells coated with bovine type I collagen. Download figure Download PowerPoint We have identified a two-domain subregion of CNA that binds collagen with high affinity, crystallized this subregion and solved its crystal structure both as an apo-protein and in complex with a synthetic, collagen-like triple-helical peptide. Analyses of these structures point to an extraordinary multistep binding mechanism where the two subdomains cooperate to wrap around and ‘hug’ the rope-like structure of a collagen monomer. The proposed binding mechanism, with some aspects of the Dock, Lock and Latch mechanism previously reported for MSCRAMM binding of linear peptides (Ponnuraj et al, 2003), demonstrates how bacteria by using similar building blocks, albeit with subtle modifications, can generate high affinity binding proteins that are specifically designed to adhere structurally diverse ligands. The possibility that eukaryotic multidomain collagen-binding proteins use similar ‘hugging’ mechanisms to embrace the collagen triple helix is also discussed. Results and discussion A CNA segment with high affinity for collagen We previously reported that the fibrinogen binding staphylococcal MSCRAMMs contain three subdomains called N1, N2 and N3 (Perkins et al, 2001; Ponnuraj et al, 2003) in their ligand-binding regions. By comparing the amino-acid sequences of CNA with these fibrinogen-binding proteins, we propose that the A-region of CNA is also composed of three subdomains; N1 corresponding to residues 31–140, N2 to residues 141–344 and N3 to residues 345–531. The previously identified minimal collagen-binding domain of CNA (res. 151–318) would, in this model, correspond to a truncated form of the N2 domain. The affinity of CNA151–318 for type I collagen is about 10-fold lower than that of the full-length CNA A-region (Patti et al, 1993). In our attempts to identify a subsegment of the CNA A-region with full binding activity, we constructed a set of seven recombinant proteins (Figure 1A, Supplementary Table 1) based on the putative subdomains and determined their relative affinity for type 1 collagen by surface plasmon resonance (SPR). Each protein at 10 μM was run over a BIA-core chip containing immobilized collagen (Figure 1B). Remarkably, the protein construct corresponding to the predicted N1N2 domains (CNA31–344) bound collagen with an affinity that appears to be substantially higher than that of even the full-length A-region (CNA31–531). Recombinant proteins corresponding to the predicted N2 domain (CNA141–344) and the N2N3 domains (CNA141–531) bound collagen with affinities similar to that of the previously examined CNA151–318, lower than that of CNA31–531 and much lower than that of CNA31–344. Recombinant proteins representing the predicted independent N1 or N3 domains did not show any measurable affinity for collagen. Further analyses using solid phase ELISA-type binding assays (Table I) or SPR analyses (data not shown) with multiple concentrations of CNA proteins confirmed the relative affinities indicated in Figure 1B. We further characterized the binding of the different recombinant CNA proteins to type 1 collagen in inhibition-ELISAs. Each of the CNA31–344, CNA31–531 and CNA151–318 proteins could completely inhibit the binding of biotin derivatives of the individual proteins to type I collagen (e.g., see Figure 1C), suggesting that the different CNA constructs have the same binding specificity but differ in their binding affinity. Table 1. Summary of the apparent collagen binding affinities of the different recombinant fragments of CNA Construct KDapp (μM)a CNA31–531 2.2±0.4 CNA31–344 0.2±0.02 CNA31–318 1.5±0.5 CNA151–318 31.6±4.4 CNA141–344 69.6±6.1 CNA151–531 28.3±3.3 CNA141–531 38.1±11.8 CNA31–156 NDb CNA319–531 NDb a The apparent dissociation constants (KDapp) were determined from ELISA assays as described in Materials and methods. Recombinant fragments of the different constructs were added to wells coated with bovine type I collagen. Bound proteins were detected with mouse anti-His antibodies and goat-anti-mouse IgG-AP conjugants. Data analysis was performed using the nonlinear regression method (GraphPad Prism). b CNA31–156 and CNA319–531 did not show any binding in the ELISA assays and their KDapp values were not determined. Identification of a collagen peptide with high affinity for CNA31–344 To identify binding sites in collagen for CNA31–344, we initially examined the binding of this MSCRAMM to fragments of bovine type I collagen generated by cyanogen bromide cleavage. CNA31–344 bound all fragments tested but with varying apparent affinities (P Speziale, Y Xu and M Höök, unpublished observation). Rotary shadowing coupled with electron microscopy was used to locate CNA binding sites; however, we could not identify any particularly ‘hot spots’ and the binding sites appeared scattered over the type I collagen molecule (D Keene, Y Xu, and M Höök, unpublished observation). It seems that CNA31–344 can bind many sites along the collagen molecule, in agreement with previous SPR analysis of the binding characteristics of CNA31–531 (Rich et al, 1999b). We screened a panel of 16 synthetic collagen peptides that were generated based on sequences from the α1 chain of bovine or chicken type I collagen. To facilitate the formation of collagen-like triple helices, the specific sequences in individual peptides were flanked by three or four GPO (O being Hydroxyproline) triplets at either side. In addition to this available library, we synthesized two generic peptides consisting of 11 GPO triplets or 11 GPP triplets. In SPR binding analysis, some of the collagen peptides exhibited a high affinity for CNA31–344, some had an intermediate affinity, and others had a low affinity. An example of the binding of four of the peptides, DBS4, (GPO)11, (GPP)11 and DBS3 are shown in Figure 2A. The CD spectra of these peptides indicated that DBS4, (GPO)11, and (GPP)11 formed triple helices at both 4°C and room temperature, while DBS3 does not (Supplementary Figure 1). The binding of DBS4, (GPO)11, and (GPP)11 to CNA31–344 was further analyzed using SPR by passing increasing concentrations of the three peptides over a surface with immobilized CNA31–344. The dissociation constants (KD) of the interactions between CNA and DBS4, (GPO)11 and (GPP)11 were determined to be ∼3.0, ∼140, and ∼7.5 nM, respectively. In solid phase inhibition assays, DBS4 and (GPP)11 completely inhibited the binding of CNA31–344 to type I collagen with IC50 values of 5.3±1.3 and 11.8±1.5 μM, respectively. (GPO)11 and DBS3 were less potent inhibitors and only caused ∼50% inhibition at 100 μM (Figure 2B). Taken together, these results suggest that DBS4 and (GPP)11 contain at least one high-affinity binding site for CNA31–344. Figure 2.(A) Representative sensorgrams of the synthetic collagen peptides passed over CNA31–344. The same concentrations of four collagen peptides (1 μM) were passed over a CNA31–344-coated surface. Injection started at ∼95 s and ended at ∼335 s. Responses from a blank surface were subtracted from responses from collagen-coated surface. (B) Inhibition of the binding of CNA31–344 to type I collagen by synthetic collagen peptides. CNA31–344 (10 nM) was preincubated with increasing amounts of synthetic collagen peptides DBS4 (•), DBS3 (▴), (GPO)11 (▾), and (GPP)11 (⧫), and then added to wells coated with bovine type I collagen. Download figure Download PowerPoint Crystal structure of apo-CNA31–344 The crystal structure of CNA31–344 was determined by molecular replacement methods, using the CNA151–318 crystal structure (Symersky et al, 1997) as a search model, and refined to an R factor of 19.1% (Rfree of 23.3%) using diffraction data to 1.95 Å resolution. Crystallographic, diffraction data collection, and model refinement details are presented in Table II. The crystal structure of CNA31–344 (Figure 3A) exhibits two distinct domains (called N1 and N2). The N-terminal N1 domain (residues 31–163) exhibits a DeV-IgG fold, which contains two additional strands D1′ and D1″ (Figure 3B) compared to the conventional IgG fold (Deivanayagam et al, 2002; Ponnuraj et al, 2003). The N2 domain corresponding to 174–329 residues is very similar to the previously determined crystal structure of CNA151–318 (Symersky et al, 1997), which contains in addition to the D2′ and D2″ strands, an extra D2‴ strand and a two-turn α-helix (Figure 3B). Figure 3.Crystal structure of the apo-CNA31–344. (A) Apo-CNA31–-344 consists two domains, N1 (Green) and N2 (Yellow). Residues 164–173 form a linker (blue in color) that joins the N1 and N2 domains. (B) Cartoon representation of the apo-CNA31–344 crystal structure. Strands are represented as arrows (green for the N1 domain and yellow for the N2 domain), and helices in blue color. The linker joining the N1 and N2 domains is represented in blue and all other loop regions in black. Images of the molecular structures were prepared using RIBBONS program (Carson, 1997). (C) The inter domain hole in apo-CNA31–344 is formed by the linker that connects N1 and N2 domains and hydrophobic residues contributed by both N1 and N2 domains. (D) Comparison of apo-CNA31–344 and SdrGN2N3 crystal structures. The crystal structure of apo-CNA31–344 (top) is compared to the ligand bound SdrGN2N3 crystal structure (bottom) to illustrate the common C-terminal extension ‘donor strand’ mode and the length differences in the inter domain linker region (blue). Download figure Download PowerPoint Table 2. Data collection and refinement statistics Crystal CNA31–344 CNA31–344–collagen peptide complex Unit cell dimensions a (Å) 41.98 90.55 b (Å) 106.43 193.82 c (Å) 44.08 205.19 β (deg) 116.45 Space group P2 (1) C222 (1) Resolution limits (Å) 1.95–50.0 3.30–50.0 Total reflections 159 629 289 690 Unique reflections 19 783 24 313 Completeness (%)a 95.8 (93.6) 94.3 (83.1) Rsymm (%)a 3.0 (6.1) 16.8 (60.7) a 36.0 (19.5) 3.5 (1.2) Refinement R 19.11 25.10 Rfree 23.33 30.21 R.m.s. deviation Bond length (Å) 0.0051 0.0101 Bond angle (deg) 1.220 1.825 Water molecules 230 0 Ramachandran plot Core 93.5% 71.3% Allowed regions 4.5% 25.5% Generously allowed 2.0% 2.5% Disallowed regions 0.0% 0.6% Over all B-factor 24.2% 39.4 R.m.s. deviation Main chain bond B values 0.98 1.75 Main chain angle B values 1.35 2.81 Side chain bond B values 2.62 3.93 Side chain angle B values 4.82 6.10 a Numbers in parentheses refer to the highest resolution shell. The N1 and N2 domains of CNA are connected by a long linker region (residues 164–173; colored blue in Figures 3A and B). This organization creates a two-domain structure with a distinct hole between the two domains (Figure 3D). The electron density for the main chain atoms of the 164–173 linker is poorly defined suggesting some flexibility in this linker segment. The N2 domain ends at residue 318 and the C-terminal extension of this domain (residues 319–329) stretches towards the N1 domain and forms a β strand (G′) that complements one of the β-sheets of the N1 domain (Figure 3A), while the following residues 332–344 are disordered and extend into the solvent region. This intra-molecular donor strand observed in CNA31–344 is analogous to the ‘latch’ observed in the crystal structure of SdrGN2N3 in complex with a ligand peptide (Ponnuraj et al, 2003) (Figure 3D). In the structure of the apo-form of SdrGN2N3, the C-terminal ‘latch’ points to the solvent, resulting in an open conformation that allows the ligand to dock onto its binding site. Upon ligand binding, due to hydrophobic interactions with the ligand peptide, the C-terminal extension of the SdrG N3 subdomain is redirected and latched into a trench present in the N2 subdomain, resulting in a closed conformation. However, in the apo-CNA31–344 structure, a closed conformation is observed with the analogous latch (residues 319–330) inserted into its N1 domain. The back of the latching trench on N1 contains a TYTFTDYVD motif, which is found in a number of MSCRAMMs (McCrea et al, 2000) and was predicted to contribute to the structuring of the ‘latching cleft’ in SdrG (Ponnuraj et al, 2003). It is interesting to note that the inter-domain N2 C-terminal extension is only three residues (319–321) long in CNA31–344, compared to six residue length of N3 C-terminal extension in SdrGN2N3, resulting in a close contact between N1 and N2 domains of CNA. The two CNA31–344 domains interact through hydrophobic residues Tyr88 and Pro108 of N1, Pro182 and Met180 of N2, and Ile319 of the C-terminal N2 extension (Figure 3C). In this closed conformation, the combined buried surface area between the N1 and N2 domains of CNA31–344 is 1493 Å2. The inter-domain N2 C-terminal extension and the hydrophobic residues present between the two domains complete the hole created by the linker 164–173 (Figure 3D) in the apo-CNA31–344 crystal structure. The overall structure of CNA31–344 in complex with a collagen peptide Crystals of CNA31–344 in complex with DBS4 [(GPO)4GPRGRT(GPO)4], a synthetic triple-helical collagen peptide, belong to the C2221 space group and diffracted anisotropically to 3.2 Å resolution at best, and 3.5 Å at worst. The asymmetric unit contains two copies of complex, each one composed of two molecules of CNA31–344 bound to one collagen-like peptide. The crystal structure of this complex was determined by molecular replacement methods using the apo-CNA31–344 crystal structure as a search model. Electron density for the collagen peptide developed gradually, and the collagen peptide model was not included in phase and map calculations until the final cycles of refinement (Figure 4A). The final model was refined to an R factor of 26.5% (Rfree of 33.5%) using diffraction data to 3.3 Å resolution. The two complex molecules present in one asymmetric unit are not related by a two-fold noncrystallographic symmetry. Figure 4.The crystal structure of (CNA31–344)2–collagen-like peptide complex. (A) The representative simulated annealing omit map for the collagen-like peptide is viewed at 1.5σ. (B) The triple helical collagen peptide is seen parked in the hole between N1 and N2 domains. The three chains of the collagen peptide are named as leading (magenta), middle (cyan) and trailing (white) as viewed from their N-termini. (C) Two CNA31–344 molecules interact with one collagen peptide in an identical manner. The leading and trailing chains peptide interact with N2 domain β sheet, and the middle chain with N1. The N1–N2 linker covers the leading and trailing chains, and holds the rope like ligand in place. Download figure Download PowerPoint The structure of the collagen-like peptide Figures 4B and C present a schematic representation of interactions between the CNA31–344 molecules and the collagen peptide. The three (GPO)4GPRGRT(GPO)4 strands form a right handed triple helix that is about 90 Å long. We named the three chains of the collagen peptide as leading, middle and trailing when viewed from their N-termini (Emsley et al, 2000). The conformation and structure of the collagen triple helix (Figure 4C) in this structure is similar to that observed for the collagen peptide (POG)4POA(POG)5 reported in 1994 (Bella et al, 1994). No structural water molecules are identified due to the low resolution of the present structure. However, the inter chain hydrogen bonds essential for the stabilization of the supercoiled structure are present similar to those observed in the (POG)4POA(POG)5 triple helical peptide. The N- and C-terminal tips of the collagen peptide are disordered and no electron density is seen for either of the termini. The peptide is slightly bent before and after the central RGRT sequence. The side chains of Arg and Thr residues in the middle of the collagen peptide are pointing to the solvent region and do not participate in intra-collagen interactions. Different chains have specific interactions with different parts of CNA31–344 in the (CNA31–344)2–collagen peptide complex, which we will discuss below. The structure of (CNA31–344)2–collagen peptide complex The crystal structure of the (CNA31–344)2–collagen peptide complex looks like a dumbbell, with two CNA31–344 molecules bound at each end of the collagen peptide (Figure 4C). The collagen triple helix is seen penetrating through the hole between the N1 and N2 domains in CNA31–344 (Figure 4B). This hole is about 12 to 15 Å in diameter. The overall diameter of the (GPO)4 region of the collagen peptide is about 11 Å, increasing to 15–19 Å around the central RGRT sequence and at termini. The GPO repeating regions of the collagen peptide precisely fit into this inter-domain hole and there is no extra space to accommodate any larger side chains (Figure 5A). Figure 5.(A) A surface plot of the collagen peptide with the two CNA31–344 molecules. The N1–N2 linker regions are shown in blue. (B) Two CNA31–344 molecules in one complex molecule have a rotational difference of 120° when viewed down the collagen peptide from N-terminus. (C) The conformational switch of a loop region (residue 138–148) between apo-CNA31–344(green) and ligand bound CNA31–344 (yellow) for stabilizing the bound ligand. Download figure Download PowerPoint In the crystal structure, a turn in the super coil of the collagen is calculated to require approximately 7.3 GXY (X and Y represent any amino acids) repeats, giving an average 49° rotational difference between the consecutive GXY repeats. However, the helical pitch between the GPO repeats varies among the various regions of the (GPO)4GPRGRT(GPO)4 peptide, suggesting a significant helical twist relaxation, which may be the result of various amino acid substitutions in the X and Y positions (Bella et al, 1994). Hence, the approximate 75 triple-helical symmetry observed for (GPO)4GPRGRT(GPO)4 is different from the ideal 107 symmetry calculated from fiber diffraction experiments of native collagen (Fraser et al, 1979; Okuyama et al, 1981). The first GPO repeat that is bound by one CNA31–344 molecule is five repeats apart from the first GPO repeat bound by the second CNA31–344 molecule. The rotational difference between the two CNA31–344 molecules is around −245° (115°; Figure 5C), close to the expected 255° value for five repeats in a collagen peptide with 75 screw symmetry. Viewing the collagen peptide from the
Referência(s)