Vengadesan Krishnan, Andrew H. Gaspar, Naiqing Ye, Anjali Mandlik, Hung Ton-That, and Narayana S. V. L (2007) An IgG-like Domain in the Minor Pilin GBS52 of Streptococcus agalactiae Mediates Lung Epithelial Cell Adhesion. Structure 15(8):893-903 |
Streptococcus agalactiae is the leading cause of neonatal pneumonia, sepsis, and meningitis. The pathogen assembles heterotrimeric pilus structures on its surface; however, their function in pathogenesis is poorly understood. We report here the crystal structure of the pilin GBS52, which reveals two IgG-like fold domains, N1 and N2. Each domain is comprised of seven antiparallel beta strands, an arrangement similar to the fold observed in the Staphylococcus aureus adhesin Cna. Consistent with its role as an adhesin, deletion of gbs52 gene significantly reduces bacterial adherence to pulmonary epithelial cells. Moreover, latex beads linked to the GBS52 protein adhere to pulmonary but not to many other epithelial cells; binding to the former is specifically inhibited by antibodies against GBS52. Nonetheless, substantial binding is only observed with N2 domain-conjugated beads. This study presents the structure of a Gram-positive pilin that utilizes a distinct IgG fold variant to mediate pathogen adherence to a specific tissue.
Liu Q, Ponnuraj K, Xu Y, Ganesh V, Sillanpaa J, Murray BE, Narayana, S.V.L, Hook M. (2007) The enterococcus faecalis MSCRAMM ACE binds its ligand by the collagen hug model. J. Biol. Chem (In Print)
We have determined the crystal structure of the ligand binding segment of the Enterococcus faecalis collagen binding MSCRAMM ACE. This segment is composed of two subdomains, N1 and N2, each adopting an IgG-like fold and forming a putative collagen binding surface at the interphase between the two subdomains. This structure is very similar to that recently reported for CNA, the collagen binding MSCRAMM of Staphylococcus aureus, for which a unique ligand binding mechanism called the Collagen Hug was proposed. We suggest that ACE binds collagen by a similar mechanism and present the first biochemical evidence for this binding model. Mutations of ACE by replacing residues in the putative collagen binding trench of N2 with Ala residues affected collagen binding. A closed conformation of ACE stabilized by an engineered disulfide bond is predicted and demonstrated to be unable to bind collagen. Finally the importance of the residues in the N2 extension in stabilizing the MSCRAMM-ligand complex is demonstrated by selected point and truncation mutations.
Vengadesan Krishnan, Yuanyuan Xu, Kevin Macon, John E. Volanakis and Narayana, S. V. L. (2007) “The Crystal Structure of C2a, the Catalytic Fragment of Classical Pathway C3- and C5-convertase of Human Complement”. J. Mol. Biol. 367, 224-233.
The multi-domain serine protease C2 provides the catalytic activity for the C3 and C5-convertases of the classical and lectin pathways of complement activation. Formation of these convertases requires the Mg(2+)-dependent binding of C2 to C4b, and the subsequent cleavage of C2 by C1s or MASP2, respectively. The C-terminal fragment C2a consisting of a serine protease (SP) and a von Willebrand factor type A (vWFA) domain, remains attached to C4b, forming the C3-convertase, C4b2a. Here, we present the crystal structure of Mg(2+)-bound C2a to 1.9 A resolution in comparison to its homolog Bb, the catalytic subunit of the alternative pathway C3-convertase, C3bBb. Although the overall domain arrangement of C2a is similar to Bb, there are certain structural differences. Unexpectedly, the conformation of the metal ion-dependent adhesion site and the position of the alpha7 helix of the vWFA domain indicate a co-factor-bound or open conformation. The active site of the SP domain is in a zymogen-like inactive conformation. On the basis of these structural features, we suggest a model for the initial steps of C3-convertase assembly.
Ponnuraj, K and Narayana, S. V. L (2007) “Crystal structure of ACE19, the collagen binding subdomain of Enterococus faecalis surface protein ACE” Proteins (In print).
Ajees, A.A, Gunasekaran, K., Volanakis, J. E., Narayana, S. V. L., Kotwal, G. J., and Murthy, H. M. K (2006) “The Structure of Complement C3b: Insights into Complement Activation and Regulation” Nature ; 444, :221-225.
The human complement system is an important component of innate immunity. Complement-derived products mediate functions contributing to pathogen killing and elimination. However, inappropriate activation of the system contributes to the pathogenesis of immunological and inflammatory diseases. Complement component 3 (C3) occupies a central position because of the manifold biological activities of its activation fragments, including the major fragment, C3b, which anchors the assembly of convertases effecting C3 and C5 activation. C3 is converted to C3b by proteolysis of its anaphylatoxin domain, by either of two C3 convertases. This activates a stable thioester bond, leading to the covalent attachment of C3b to cell-surface or protein-surface hydroxyl groups through transesterification. The cleavage and activation of C3 exposes binding sites for factors B, H and I, properdin, decay accelerating factor (DAF, CD55), membrane cofactor protein (MCP, CD46), complement receptor 1 (CR1, CD35) and viral molecules such as vaccinia virus complement-control protein. C3b associates with these molecules in different configurations and forms complexes mediating the activation, amplification and regulation of the complement response. Structures of C3 and C3c, a fragment derived from the proteolysis of C3b, have revealed a domain configuration, including six macroglobulin domains (MG1-MG6; nomenclature follows ref. 5) arranged in a ring, termed the beta-ring. However, because neither C3 nor C3c is active in complement activation and regulation, questions about function can be answered only through direct observations on C3b. Here we present a structure of C3b that reveals a marked loss of secondary structure in the CUB (for 'complement C1r/C1s, Uegf, Bmp1') domain, which together with the resulting translocation of the thioester domain provides a molecular basis for conformational changes accompanying the conversion of C3 to C3b. The total conformational changes make many proposed ligand-binding sites more accessible and create a cavity that shields target peptide bonds from access by factor I. A covalently bound N-acetyl-l-threonine residue demonstrates the geometry of C3b attachment to surface hydroxyl groups.
Zong, Y., Xu,Y., Gurusiddappa, S., Hook, M and Narayana, S. V. L. (2005) A ‘Collagen Hug’ model for S. Aureus MSCRAMM CNA binding to collagen” EMBO Journal, 24, 4224-4236.
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 sub-segment 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 beta-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.
Zong, Y., Bice, T., Ton-That, H., Schneewind, O., and Narayana, S. V. L. (2004) Crystal structures of sortase A and its substrate complex.
J. Biol. Chem. 279, 31383-31389.
The cell wall envelope of staphylococci and other Gram-positive pathogens is coated with surface proteins that interact with human host tissues. Surface proteins of Staphylococcus aureus are covalently linked to the cell wall envelope by a mechanism requiring C-terminal sorting signals with an LPXTG motif. Sortase (SrtA) cleaves surface proteins between the threonine (T) and the glycine (G) of the LPXTG motif and catalyzes the formation of an amide bond between threonine at the C-terminal end of polypeptides and cell wall cross-bridges. The active site architecture and catalytic mechanism of sortase A has hitherto not been revealed. Here we present the crystal structures of native SrtA, of an active site mutant of SrtA, and of the mutant SrtA complexed with its substrate LPETG peptide and describe the substrate binding pocket of the enzyme. Highly conserved proline (P) and threonine (T) residues of the LPXTG motif are held in position by hydrophobic contacts, whereas the glutamic acid residue (E) at the X position points out into the solvent. The scissile T-G peptide bond is positioned between the active site Cys(184) and Arg(197) residues and at a greater distance from the imidazolium side chain of His(120). All three residues, His(120), Cys(184), and Arg(197), are conserved in sortase enzymes from Gram-positive bacteria. Comparison of the active sites of S. aureus sortase A and sortase B provides insight into substrate specificity and suggests a universal sortase-catalyzed mechanism of bacterial surface protein anchoring in Gram-positive bacteria.
Marrraffini, L. A., Ton-That, H., Zong, Y., Narayana, S. V. L., and Schneewind, O. (2004) Anchoring of surface proteins to the cell wall of Staphylococcus aureus, J. Biol. Chem. 279, 37763-37770.
Surface proteins of Staphylococcus aureus are anchored to the cell wall envelope by a mechanism requiring a C-terminal sorting signal with an LPXTG motif. Sortase A cleaves surface proteins between the threonine (T) and the glycine (G) residues of the LPXTG motif and catalyzes the formation of an amide bond between the carboxyl group of threonine at the C-terminal end of polypeptides and the amino group of pentaglycine cross-bridges of cell wall peptidoglycan. Previous work showed that Cys(184) and His(120) of sortase A are absolutely essential for catalysis; however an active site thiolateimidazolium ion pair may not be formed. The three-dimensional crystal structure of sortase A revealed that Arg(197) is located in close proximity to both the active site Cys(184) and the scissile peptide bond between threonine and glycine. We show here that substitution of Arg(197) with alanine, lysine, or histidine severely reduced sortase A function both in vivo and in vitro, whereas Asn(98), which had earlier been implicated in hydrogen bonding to His(120), was found to be dispensable for catalysis. As the structural proximity of Arg(197) and Cys(184) is conserved in sortase enzymes and as ionization of the Cys(184) sulfhydryl group seems required for sortase activity, we propose that Arg(197) may function as a base, facilitating thiolate formation during sortase-mediated cleavage and transpeptidation reactions.
Xu, Y., Ponnuraj, K., Narayana, S. V. L., and Volanakis, J. E. (2004) Factor D and Factor B. substrate-Inducible serine proteases of the alternative pathway of complement activation. In Structural Biology of the Complement system. Eds Lambris and Morikis. Marcel Dekkev Inc., 91-110.
Ponnuraj, K., Xu, Y., Macon, K., Moore, D., Volanakis, J., and Narayana, S. V. L. (2004) “Structural Analysis of Engineered Bb Fragment of Complement Factor B: Insights into the Activation Mechanism of the Alternative Pathway C3-Convertase. Molecular Cell, 14, 17-28.
The C-terminal fragment, Bb, of factor B combines with C3b to form the pivotal C3-convertase, C3bBb, of alternative complement pathway. Bb consists of a von Willebrand factor type A (vWFA) domain that is structurally similar to the I domains of integrins and a serine protease (SP) domain that is in inactive conformation. The structure of the C3bBb complex would be important in deciphering the activation mechanism of the SP domain. However, C3bBb is labile and not amenable to X-ray diffraction studies. We engineered a disulfide bond in the vWFA domain of Bb homologous to that shown to lock I domains in active conformation. The crystal structures of Bb(C428-C435) and its inhibitor complexes reveal that the adoption of the "active" conformation by the vWFA domain is not sufficient to activate the C3-convertase catalytic apparatus and also provide insights into the possible mode of C3-convertase activation.
Zong, Y., Mazamenian, S., Schneewind, O., and Narayana S. V. L (2004) Structural basis for surface protein anchoring in S. Aureus: Sortase B is a unique cysteine transpeptidase that utilizes CYS-ARG catalytic dyad, Structure 12, 115-112.
Many surface proteins of Gram-positive bacteria, which play important roles during the pathogenesis of human infections, are anchored to the cell wall envelope by a mechanism requiring sortases. Sortase B, a cysteine transpeptidase from Staphylococcus aureus, cleaves the C-terminal sorting signal of IsdC at the NPQTN motif and tethers the polypeptide to the pentaglycine cell wall cross-bridge. During catalysis, the active site cysteine of sortase and the cleaved substrate form an acyl intermediate, which is then resolved by the amino group of pentaglycine cross-bridges. We report here the crystal structures of SrtB(DeltaN30) in complex with two active site inhibitors, MTSET and E64, and with the cell wall substrate analog tripleglycine. These structures reveal, for the first time, the active site disposition and the unique Cys-Arg catalytic machinery of the cysteine transpeptidase, and they also provide useful information for the future design of anti-infective agents against sortases.
Ponnuraj, K., Bowden, M, G., Davis, S., Gurusiddappa, S., Moore, D., Choe, C., Hook, M., and Narayana, S. V. L. (2003) A “dock, lock and latch” Structural Model for a Staphylococcal Adhesin Binding to Fibrinogen.
Cell 115, 217-228.
Gram-positive pathogens such as staphylococci contain multiple cell wall-anchored proteins that serve as an interface between the microbe and its environment. Some of these proteins act as adhesins and mediate bacterial attachment to host tissues. SdrG is a cell wall-anchored adhesin from Staphylococcus epidermidis that binds to the Bbeta chain of human fibrinogen (Fg) and is necessary and sufficient for bacterial attachment to Fg-coated biomaterials. Here, we present the crystal structures of the ligand binding region of SdrG as an apoprotein and in complex with a synthetic peptide analogous to its binding site in Fg. Analysis of the crystal structures, along with mutational studies of both the protein and of the peptide, reveals that SdrG binds to its ligand with a dynamic "dock, lock, and latch" mechanism. We propose that this mechanism represents a general mode of ligand binding for structurally related cell wall-anchored proteins of gram-positive bacteria.
Todd, B., Moore, D., Deivanayagam, C. S. S., Lin, G., Chattopadhyay, D., Maki, M., Wang, K. K. W., and Narayana, S. V. L (2003) A structural Model for the inhibition of calpain by calpastatin: Crystal Structure of the Native Domain VI of Calpain and its Complexes with Calpastatin Peptide and small molecule Inhibitor. J. Mol. Biol.. 328,131-146.
The Ca(2+)-dependent cysteine protease Calpain along with its endogenous inhibitor Calpastatin is widely distributed. The interactions between Calpain and Calpastatin have been studied to better understand the nature of Calpain inhibition by calpastatin, which can aid the design of small molecule inhibitors to Calpain. Here we present the crystal structure of a complex between a Calpastatin peptide and the calcium-binding domain VI of calpain. DIC19 is a 19 residue peptide, which corresponds to one of the three interacting domains of Calpastatin, which is known to interact with domain VI of Calpain. We present two crystal structures of DIC19 bound to domain VI of Calpain, determined by molecular replacement methods to 2.5A and 2.2A resolution. In the process of crystallizing the inhibitor complex, a new native crystal form was identified which had the homodimer 2-fold axis along a crystallographic axis as opposed to the previously observed dimer in the asymmetric unit. The crystal structures of the native domain VI and its inhibitor PD150606 (3-(4-iodophenyl)-2-mercapto-(Z)-2-propenoic acid) complex were determined with the help of molecular replacement methods to 2.0A and 2.3A resolution, respectively. In addition, we built a homology model for the complex between domain IV and DIA19 peptide of Calpastatin. Finally, we present a model for the Calpastatin-inhibited Calpain.
Deivanayagam, C. C. S., Wann, E. R., Chen, W., Carson, M., Rajashankar, K. R.,
Hook, M, Narayana, S. V. L. (2002) A Novel variant of the immunoglobulin fold in surface adhesins of Staphylococcus aureus: Crystal structure of the fibrinogen binding MSCRAMM, Clumping factor A. EMBO Journal, 21, 6660-667.
We report here the crystal structure of the minimal ligand-binding segment of the Staphylococcus aureus MSCRAMM, clumping factor A. This fibrinogen-binding segment contains two similarly folded domains. The fold observed is a new variant of the immunoglobulin motif that we have called DE-variant or the DEv-IgG fold. This subgroup includes the ligand-binding domain of the collagen-binding S.aureus MSCRAMM CNA, and many other structures previously classified as jelly rolls. Structure predictions suggest that the four fibrinogen-binding S.aureus MSCRAMMs identified so far would also contain the same DEv-IgG fold. A systematic docking search using the C-terminal region of the fibrinogen gamma-chain as a probe suggested that a hydrophobic pocket formed between the two DEv-IgG domains of the clumping factor as the ligand-binding site. Mutagenic substitution of residues Tyr256, Pro336, Tyr338 and Lys389 in the clumping factor, which are proposed to contact the terminal residues (408)AGDV(411) of the gamma-chain, resulted in proteins with no or markedly reduced affinity for fibrinogen.
Ponnuraj, K, Xu, Y., Moore, D., Deivanayagam, C. C. S., Boque, L.,Hook, M., and Narayana, S. V. L. (2002) Crystallization and Preliminary X-ray Crystallographic analysis of Ace: a collagen binding MSCRAMM from E. faecalis.” BioChem. Biophys. Acta, 173-176
Perkins, S., Walsh, E. J., Deivanayagam, C. S., Narayana, C. S., Foster, T. J., and Hook, M. (2001) Structural organization of the fibrinogen-binding region of the clumping factor B MSCRAMM of Staphylococcus aureus.
J. Biol. Chem, 276, 44721-44728.
Xu, Y., Narayana, S. V. L., and Volanakis, J. E. (2001) Structural Biology of the alternative pathway convertase. Immunological Reviews, 180, 123-135
Narayana, S. V. L., Babu, Y. S., and Volanakis, J. E. (2000) Inhibition of complement serine protease's In ‘Therapeutic Interventions in the Complement System. Ed. Lambris, J. D., and Holers, V. M. Humana Press.
Xu, Y., Circolo, A., Jing, H., Wang, Y., Narayana, S. V. L. and Volanakis, J. E. (2000) Mutational analysis of the primary substrate specificity pocket of complement factor B: Asp226 is a major structural determinant for P1-Arg binding. J. Biol. Chem. 275, 378-385.
Factor B is a serine protease, which despite its trypsin-like specificity has Asn instead of the typical Asp at the bottom of the S(1) pocket (position 189, chymotrypsinogen numbering). Asp residues are present at positions 187 and 226 and either one could conceivably provide the negative charge for binding the P(1)-Arg of the substrate. Determination of the crystal structure of the factor B serine protease domain has revealed that the side chain of Asp(226) is within the S(1) pocket, whereas Asp(187) is located outside the pocket. To investigate the possible role of these atypical structural features in substrate binding and catalysis, we constructed a panel of mutants of these residues. Replacement of Asp(187) caused moderate (50-60%) decrease in hemolytic activity, compared with wild type factor B, whereas replacement of Asn(189) resulted in more profound reductions (71-95%). Substitutions at these two positions did not significantly affect assembly of the alternative pathway C3 convertase. In contrast, elimination of the negative charge from Asp(226) completely abrogated hemolytic activity and also affected formation of the C3 convertase. Kinetic analyses of the hydrolysis of a P(1)-Arg containing thioester by selected mutants confirmed that residue Asp(226) is a primary structural determinant for P(1)-Arg binding and catalysis
Jing, H., Xu, Y., Carson, M., Circolo, A., Moore, D., Macon, K. J., Delucas, , L. J., Volanakis, J. E. and Narayana, S. V. L. (2000) New structural motifs of the chymotrypsin fold and their potential roles in complement factor B.
EMBO journal, 19, 164-173.
Factor B and C2 are two central enzymes for complement activation. They are multidomain serine proteases and require cofactor binding for full expression of proteolytic activities. We present a 2.1 A crystal structure of the serine protease domain of factor B. It shows a number of structural motifs novel to the chymotrypsin fold, which by sequence homology are probably present in C2 as well. These motifs distribute characteristically on the protein surface. Six loops surround the active site, four of which shape substrate-binding pockets. Three loops next to the oxyanion hole, which typically mediate zymogen activation, are much shorter or absent. Three insertions including the linker to the preceding domain bulge from the side opposite to the active site. The catalytic triad and non-specific substrate-binding site display active conformations, but the oxyanion hole displays a zymogen-like conformation. The bottom of the S1 pocket has a negative charge at residue 226 instead of the typical 189 position. These unique structural features may play different roles in domain-domain interaction, cofactor binding and substrate binding.
Deivanayagam, C. S. S., Rich, R. L., Carson, M., Owens, R. T., Danthuluri, S., Bice, T., Hook, M. and Narayana, S. V. L. (2000) Novel fold and assembly of the repetitive B region of the Staphylococcus aureus collagen-binding surface protein. Structure, 8, 67-78.
The Staphylococcus aureus collagen-binding protein Cna mediates bacterial adherence to collagen. The primary sequence of Cna has a non-repetitive collagen-binding A region, followed by the repetitive B region. The B region has one to four 23 kDa repeat units (B(1)-B(4)), depending on the strain of origin. The affinity of the A region for collagen is independent of the B region. However, the B repeat units have been suggested to serve as a 'stalk' that projects the A region from the bacterial surface and thus facilitate bacterial adherence to collagen. To understand the biological role of these B-region repeats we determined their three-dimensional structure. B(1) has two domains (D(1) and D(2)) placed side-by-side. D(1) and D(2) have similar secondary structure and exhibit a unique fold that resembles but is the inverse of the immunoglobulin-like (IgG-like) domains. Comparison with similar immunoglobulin superfamily (IgSF) structures shows novel packing arrangements between the D(1) and D(2) domains. In the B(1)B(2) crystal structure, an omission of a single glycine residue in the D(2)-D(3) linker loop, compared to the D(1)-D(2) and D(3)-D(4) linker loops, resulted in projection of the D(3) and D(4) in a spatially new orientation We also present a model for B(1)B(2)B(3)B(4). The B region of the Cna collagen adhesin has a novel fold that is reminiscent of but is ‘inverse’ in nature to the IgG fold. This B region assembly could effectively provide the needed flexibility and stability for presenting the ligand binding A region away from the bacterial cell surface.
Deivanayagam, C. S., Carson, M., Thotakura, A., Narayana, S. V. L., and Chodavarapu, R. S. (2000). The crystal structure of FKBP12.6 in complex with rapamycin. Acta. Cryst. D. D56, 266-271.
The crystal structure of FKBP12.6 in complex with rapamycin has now been determined at 2.0 A resolution. The structures of FKBP12.6 and FKBP12 are nearly identical, except for a displacement observed in the helical region of FKBP12.6 toward the hydrophobic pocket. This displacement was not predicted by homology modeling studies. Analyses of the residues that are likely to confer the RyR2-binding specificity are presented.
Rich, R. L., Deivanayagam, C. C. S., Owens, R. T., Carson, M., Hook, A., Moore, D., Yang, V. W., Narayana, S. V. L. and Hook, M. (1999). Trench-shaped binding sites promote multiple classes of interactions between collagen and the adherence receptors, a1b1 integrin and Staphylococcus aureus Cna MSCRAMM. J. Biol. Chem. 274, 35, 24906-24913.|
Rich, R. L., Kriekmeyer, B., Owens, R. T., LaBrenz, S., Narayana, S. V. L., Weinstock, G. M., Murry, B. E. and Hook, M. (1999) Ace Is A collagen-binding MSCRAMM from Enterococcus faecalis. J. Biol. Chem. 274, 26939-26945.
A putative collagen-binding MSCRAMM, Ace, of Enterococcus faecalis was identified by searching bacterial genome data bases for proteins containing domains homologous to the ligand-binding region of Cna, the collagen-binding MSCRAMM from Staphylococcus aureus. Ace was predicted to have a molecular mass of 71 kDa and contains features characteristic of cell surface proteins on Gram-positive bacteria, including a LPXTG motif for cross-linking to the cell wall. The N-terminal region of Ace contained a region (residues 174-319) in which 56% of the residues are identical or similar when compared with the minimal ligand-binding region of Cna (Cna 151-318); the remainder of the Ace A domain has 46% similarity with the corresponding region of the Cna A domain. Antibodies raised against recombinant Ace A domain were used to verify the cell surface expression of Ace on E. faecalis. These antibodies also effectively inhibited the adhesion of enterococcal cells to a collagen substrate, suggesting that Ace is a functional collagen-binding MSCRAMM. Structural modeling of the conserved region in Ace (residues 174-319) suggested a structure very similar to that reported for residues 151-318 of the Cna collagen-binding domain in which the ligand-binding site was identified as a trench transversing a beta-sheet face (Symersky, J., Patti, J. M., Carson, M., House-Pompeo, K., Teale, M., Moore, D., Jin, L., DeLucas, L. J., Höök, M., and Narayana, S. V. L. (1997) Nat. Struct. Biol. 10, 833-838). Biochemical analyses of recombinant Ace and Cna A domains supported the modeling data in that the secondary structures were similar as determined by CD spectroscopy and both proteins bound at multiple sites in type I collagen with micromolar affinities, but with different apparent kinetics. We conclude that Ace is a collagen-binding MSCRAMM on enterococci and is structurally and functionally related to the staphylococcal Cna protein.
Narayana, S. V. L., Elce, J. E., Chattopadhyay, D., Lin, G., Carson, M., Blanchard, H., Grouchulski, P., and Cygler, M. (1999) Structure and Assembly of the Calcium-Binding Domains of Calpain. In CALPAIN: Pharmacology and Toxicology of Calcium Dependent Protease. Eds: Wang and Yuen. Taylor and Francis.
Mayne, R., Ren, Z-X.,Liu, J., Cook, T., Carson, M., and Narayana, S. V. L. (1999) VIT-I: the second member of a new branch of the von Willebrand factor A domain super family” Bio. Soc. Tran, 27, 832-835.
Deivanayagam, C. C. S., Rich, R. L., Danthuluri, S., Owens, R. T., . Patti, J. L., Hook, M., DeLucas, L. J.,and Narayana, S. V. L. (1999). Crystallization and preliminary X-ray analysis of B-domain fragments of Staphylococcus Aureus Collagen-Binding protein. Acta. Cryst. D55, 525-527.
Deivanayagam, C. C. S, Samual Perkins, Sita Danthuluri, Rick T. Owens, Todd Bice, Tamanna Nanavathy, Timothy J. Foster, Magnus Hook, and Narayana, S. V. L. (1999) Crystallization of ClfA and ClfB fragments: the fibrinogen-binding surface proteins of Staphylococcus Aureus. Acta Cryst. D55, 554-556.
Barchue, J., Symersky, J., Narayana, S. V. L., Moore, J. K., Delucas, L. J., and Chattopadhyay, D. (1999) Expression, purification, crystallization and preliminary X-ray diffraction analysis of uracil phopphoribosyltransferase of Toxoplasma gondii. Acta Cryst D55, 347-349.
Jing, H., Macon, K., Moore, D., DeLucas, L. J., Volanakis, J.E., and Narayana, S. V. L. (1999) Structural basis of pro-factor D activation: from a highly flexible zymogen to a novel self-inhibited serine protease, complement factor D.
EMBO Journal 18, 804-814
The crystal structure of profactor D, determined at 2.1 A resolution with an Rfree and an R-factor of 25.1 and 20.4%, respectively, displays highly flexible or disordered conformation for five regions: N-22, 71-76, 143-152, 187-193 and 215-223. A comparison with the structure of its mature serine protease, complement factor D, revealed major conformational changes in the similar regions. Comparisons with the zymogen-active enzyme pairs of chymotrypsinogen, trypsinogen and prethrombin-2 showed a similar distribution of the flexible regions. However, profactor D is the most flexible of the four, and its mature enzyme displays inactive, self-inhibited active site conformation. Examination of the surface properties of the N-terminus-binding pocket indicates that Ile16 may play the initial positioning role for the N-terminus, and Leu17 probably also helps in inducing the required conformational changes. This process, perhaps shared by most chymotrypsinogen-like zymogens, is followed by a factor D-unique step, the re-orientation of an external Arg218 to an internal position for salt-bridging with Asp189, leading to the generation of the self-inhibited factor D.
Rich, R. L., Demeler, B., Deivanayagam, C. C. S., Petrich, J. W., Patti, J. M., Narayana, S. V. L., and Hook, M. (1998) Domain Structure of the Staphylococcus Aureus Collagen Adhesin. Biochemistry, 34, 14523-1543.
Arlaud, G., Volanakis, J. E., Thielens, N. M., Narayana, S. V. L., Rossi, V., and Xu, Y. (1998) The Atypical Serine Proteases of the complement system. Advances in Immunology, 69, 249-307.
Jing, H., Babu, Y. S., Moore, D., Kilpatrick, J.M., Liu, X-Yang. Volanakis, J.E., and Narayana, S. V. L (1998). Structure of Native and Complexed Complement Factor D: Implications of the Atypical His57 Conformation and Self-inhibitory Loop in the Regulation of Specific Serine Protease Activity”
. J. Mol. Biol. 282, 1061-1081.
Factor D is a serine protease essential for the activation of the alternative pathway of complement. The structures of native factor D and a complex formed with isatoic anhydride inhibitor were determined at resolution of 2.3 and 1.5 A, respectively, in an isomorphous monoclinic crystal form containing one molecule per asymmetric unit. The native structure was compared with structures determined previously in a triclinic cell containing two molecules with different active site conformations. The current structure shows greater similarity with molecule B in the triclinic cell, suggesting that this may be the dominant factor D conformation in solution. The major conformational differences with molecule A in the triclinic cell are located in four regions, three of which are close to the active site and include some of the residues shown to be critical for factor D catalytic activity. The conformational flexibility associated with these regions is proposed to provide a structural basis for the previously proposed substrate-induced reversible conformational changes in factor D. The high-resolution structure of the factor D/isatoic anhydride complex reveals the binding mode of the mechanism-based inhibitor. The higher specificity towards factor D over trypsin and thrombin is based on hydrophobic interactions between the inhibitor benzyl ring and the aliphatic side-chain of Arg218 that is salt bridged with Asp189 at the bottom of the primary specificity (S1) pocket. Comparison of factor D structural variants with other serine protease structures revealed the presence of a unique "self-inhibitory loop". This loop (214-218) dictates the resting-state conformation of factor D by (1) preventing His57 from adopting active tautomer conformation, (2) preventing the P1 to P3 residues of the substrate from forming anti-parallel beta-sheets with the non-specific substrate binding loop, and (3) blocking the accessibility of Asp189 to the positive1y charged P1 residue of the substrate. The conformational switch from resting-state to active-state can only be induced by the single macromolecular substrate, C3b-bound factor B. This self-inhibitory mechanism is highly correlated with the unique functional properties of factor D, which include high specificity toward factor B, low esterolytic activity toward synthetic substrates, and absence of regulation by zymogen and serpin-like or other natural inhibitors in blood.
• Maki, M., Narayana, S. V. L., and Hitomi, K. (1997), "A growing family of the Ca2+ binding proteins with five EF-hand motifs". Biochemical Journal, 328, 718-720.|
Lin, G., Chattopadhyay, D., Maki, M., Wang, K.K.W., Carson, M., Yuen, Takanao, E., Hatanaka, M., DeLucas, L.J., and Narayana, S. V. L. (1997), "Crystal structure of calcium bound domain VI of calpain at 1.9A resolution and its role in enzyme assembly, regulation, and inhibitor binding".
Nature Structural Biology. Vol. 4, No. 7, 539-547.
The three dimensional structure of calcium-bound domain VI of porcine calpain has been determined to 1.9 A resolution. The crystal structure reveals five EF-hands, one more than previously suggested. There are two EF-hand pairs, one pair (EF1-EF2) displays an 'open' conformation and the other (EF3-EF4) a 'closed' conformation. Unusually, a calcium atom is found at the C-terminal end of the calcium binding loop of EF4. With two additional residues in the calcium binding loop, the fifth EF-hand (EF5) is in a 'closed' conformation. EF5 pairs up with the corresponding fifth EF-hand of a non-crystallographically related molecule. Considering the EF5's role in a homodimer formation of domain VI, we suggest a model for the assembly of heterodimeric calpain. The crystal structure of a Ca2+ bound domain VI-inhibitor (PD150606) complex has been refined to 2.1 A resolution. A possible mode for calpain inhibition is discussed.
Lin, G., Chattopadhya, D., Maki, M., Takano, E., Hatanaka, M., DeLucas, L.J., and Narayana, S. V. L. (1997), "Purification, Crystallization, and Preliminary X-ray Diffraction Studies of Recombinant Calcium Binding Domain of Porcine Calpain Small Subunit". Acta. Cryst. D53, 474-476.
Moore, D. M., Bice, T., Jin, L., Asha, G., and Narayana, S. V. L. (1997), "Preliminary Crystallographic analysis of Porcine Quinolinate Phosphoribosyltransferase".Acta Cryst. D54, 119-120.
Symersky, J., Patti, J.M., Carson, M., House-Pompeo, K., Teale, M., Moore, D., Jin, L., Schneider, A., DeLucas, L.J., Hook, M., and Narayana, S. V. L. (1997), "Structure of the Collagen-binding Domain from a Staphylococcus Aureus Adhesin". Nature Structural Biology, Vol 4, No. 10, 833-838.
The crystal structure of the recombinant 19,000 M(r) binding domain from the Staphylococcus aureus collagen adhesin has been determined at 2 A resolution. The domain fold is a jelly-roll, composed of two antiparallel beta-sheets and two short alpha-helices. Triple-helical collagen model probes were used in a systematic docking search to identify the collagen-binding site. A groove on beta-sheet I exhibited the best surface complementarity to the collagen probes. This site partially overlaps with the peptide sequence previously shown to be critical for collagen binding. Recombinant proteins containing single amino acid mutations designed to disrupt the surface of the putative binding site exhibited significantly lower affinities for collagen. Here we present a structural perspective for the mode of collagen binding by a bacterial surface protein.
Agarwal, A., Lee, S., Carson, M., Narayana, S. V. L., Greenhough, T.J., Volanakis, E. (1997), "Phosphocholine-binding site of human CRP: Role of Thr76, and Trp67.” Jou. Immunol. 158, 345-350.
Cole, L. B., Chu, N., Kilpatrick, J.M., Volanakis, Narayana, S. V. L., and Babu, Y. S. (1997), "Structure of Diisopropyl Flurophosphate-Inhibited Factor D.” Acta. Cryst D53, 143-150.
Babu, Y. S., Montgomery, J. A., Bugg, C.E., Carson, W. M., Narayana, S. V. L., Cook, W. J., Ealick, S. E., Guida, W.C., Erion, M., Secrist, J.A (1997), "Design of Purine Nucleoside Phosphorylase Inhibitors". In Structure Based Inhibitor Design, Ed. Pandi Veerapandian, Mercel Dekker Inc.
Chattopadhyay, D., Stewart, J.E., Smith, C.D., DeLucas, L.J., and Narayana, S.V.L. (1997), "Preliminary Crystallographic Study on a Low Molecular Weight Form of Bacterial Plasminogen Activator, Staphylokinase".
Acta. Cryst. D53.355-356.
Narayana, S. V. L., Bugg, C.E., and Ealick, S. E. (1997), Structure of Purine Nucleoside Phosphorylase Refined at 2.75 A Resolution",
Acta. Cryst. D 53, 131-142.
Volanakis, J. E., and Narayana, S. V. L., (1996), "Factor D, a unique serine protease" Protein Science, Vol 5, No. 4, 553-564.
Factor D is unique among serine proteases in that it requires neither enzymatic cleavage for expression of proteolytic activity nor inactivation by a serpin for its control. Regulation of factor D activity is instead attained by a novel mechanism that depends on reversible conformational changes for expression and control of catalytic activity. These conformational changes are believed to be induced by the single natural substrate, C3bB, and to result in realignment of the catalytic triad, the specificity pocket, and the nonspecific substrate binding site, all of which have atypical conformations. Mutational studies have defined structural determinants responsible for these unique structural features of factor D and for the resultant low reactivity with synthetic esters.
Kim, S., Narayana, S. V. L., and Volanakis, J. E. (1995),"A crystal structure of a factor D mutant with higher catalytic activity ".
J. Biol. Chem., Vol 270, No. 41, 24399- 24405.
Kim, S., Narayana, S. V. L., and Volanakis, J. E. (1995) "Catalytic role of a surface loop of the complement serine protease factor D.”
J. Imminol.154, 6073-6079.
El-Kabbani, O. A., Green, G.N., Lin, G., Carson, M., Narayana, S.V.L., Moore, K. M., Flynn, T.G., and DeLucas, L.J. (1994), "Three-dimensional Structures of Human and Porcine Aldehyde Reductase", Acta Cryst D50, 859-868. |
Carson, M., Bugg, C.E., DeLucas, L.J., and Narayana, S. V. L. (1994), "Structure of Novel Serine Protease Compared to Homology Model",
Acta Cryst. D50, 889-899.
Carson, M., Buckner, T. W., Yang, Z., Narayana, S. V. L. and Bugg, C. E. (1994), "Error Detection in Crystallographic Models",
Acta. Cryst. D50, 900-909.
Kim, S., Narayana, S. V. L., and Volanakis, J.E. (1994), “Mutational Analysis of the Substrate Binding Site of Human Complement Factor D”, Biochemistry, 33 (48), 14393-14399.
Narayana, S. V. L., Carson, M., El-Kabbani, O. A., Kilpatrick, J. M., Moore, D., Chen, Xi, Bugg, C. E., Volanakis, J. E., and DeLucas, L. J. (1994), "Structure of Human Factor D, a Complement System Protein, at 2.0 A Resolution"
J. Mol. Biol., 235, 695-708.
Factor D, an essential enzyme for the activation of the alternative pathway of the complement system, belongs to the serine protease superfamily. The crystal structure of the enzyme was solved by a combination of multiple isomorphous replacement and molecular replacement methods. The present model was refined to an R-factor of 18.8% using 23,681 observed reflections between 7.5 and 2.0 A resolution, with a root-mean-square deviation from standard bond lengths of 0.016 A. The two non-crystallographically related molecules in the triclinic unit cell have distinctive active site conformations. The protein has the general structural fold of a serine protease, but there are several unique amino acid substitutions resulting in significant alterations in the critical loops responsible for catalysis and substrate specificity in serine proteases. Factor D is the first complement serine protease whose three-dimensional structure has been determined.
Narayana, S. V. L., Yamauchi, Y., Macon, K. M., Moore, D., DeLucas, L. J., and Volanakis, J. E. (1994), "Preliminary Crystallographic Studies on Human Complement Pro-Factor D", J. Mol. Biol., 235, 1144-1146.
DeLucas, L.J., Moore, K. M., Narayana, S. V. L., Bray, T.L., Rosenblum, W.M., Einspahr, H.M., Clancy, L.L., Rao, G.S.J., Harris, B.G., Munson, S.H., Finzel, B.C., and Bugg, C.E. (1993), "Protein Crystal Growth Results from the United States Microgravity Laboratory-1 Mission", J. of Physics D:
Applied Physics, 26, B100-B103.
DeLucas, L.J., Long, M. M., Moore, K. M., Smith, C., Carson, M., Narayana, S. V. L., Carter, D., Clark, Jr., A. D., Nanni, R.G., Ding, J., Jacobo-Molina, A., Kamer, G., Hughers, S.H., Arnold, E., Einspahr, H.M., Clancy, L.L., Rao, G.S.J., Cook, P.F., Harris, B.G., Munson, S.H., Finzel, B.C., McPherson, A., Weber, P.C., Lewandowski, F., Nagabhushan, T.L., Trotta, P.P., Reichert, P., Navia, M. A. Wilson, K.P., Thompson, J. A., Mead, C., Bishop, S. P., Unbar, B.J., Trinh, E., Prattle, J., Sac, Jr., A., and Bugg, C.E. (1993), "Recent Results and New Hardware Developments for Protein Crystal Growth in Microgravity",
J. of Crystal Growth, 135, 183-195.
El-Kabbani, O.A., Lin, G., Narayana, S. V. L., Moore, K. M., Green, N.C., Flynn, T.G., and DeLucas, L.J. (1993), "Crystallization and Preliminary Structure Determination of Porcine Aldehyde Reductase from Two Crystal Forms", Acta. Cryst. D49, 490-496.
Narayana, S. V. L., Kilpatrick, J.M., El-Kabbani, O.A., Babu, Y. S., Bugg, C.E., Volanakis, J.E., and DeLucas, L.J. (1991), "Crystallization and Preliminary X-ray Investigation of Factor D of Human Complement", J. Mol. Biol., 213, 1-3.
El-Kabbani, O. A., Narayana, S. V. L., Babu, Y. S., Moore, K. M., Flynn, T.G., Petrash, J.M., Westbrook, E. M., DeLucas, L.J., and Bugg, C.E. (1991), "Purification, Crystallization, and Preliminary Crystallographic Analysis of Porcine Aldose Reductase", J. Mol. Biol., 218, 695-698.
Carson, M., Narayana, S. V. L., DeLucas, L. J., El-Kabbani, O. A., Kilpatrick, J.M., Volanakis, J. E., and Bugg, C.E. (1991), "Modeling a Protein from Homology: Comparison with Experimental Results",
Computer Visualization, 1, 28-29.
Ealick, S.E., Babu, Y. S., Narayana, S. V. L., Cook, W.J., and Bugg, C.E. (1989), "Design of Purine Nucleoside Phosphorylase Inhibitors Using X-ray Crystallography", in Crystallographic and Modeling Methods in Molecular Design (Eds. Bugg, C.E., and Ealick, S.E.) Springer-Verlag, New York, pp. 43-55.|
Ealick, S.E., Babu, Y. S., Narayana, S. V. L., Cook, W.J., and Bugg, C.E. (1988), "Rational Drug Design for the treatment of AIDS Using X-ray Crystallography", Advances in Chemotherapy of AIDS Symposium Proceedings, Pharmacology and Therapeutics, pp. 97-106.
Cook, W.J., Koszalka, G., Hall, W.W., Narayana, S. V. L., and Ealick, S.E. (1987), "Crystallization and Preliminary X-ray Investigation of Uridine Phosphorylase from E. coli", J. Biol. Chem., 262, 2852-2853.
Deschene, R.J., Narayana, S. V. L., Argos, P. and Dixon, J.E. (1985), "Primary Structural Comparison of Preprohormones Cholecystokinin and Gastrin", FEBS Letters 182, 135-138.
Argos., P., Landy,A., Aberemski, K,……..Kalionis, B., Narayana, S. V. L., Pierson, L., Sternberg, N., and Leong, J. (1986) The integrase family of site-specific recombinase regional simil;arities and global diversity”
EMBO Journal, 5,433-440.
Argos, P., Narayana, S. V. L. and Nielsen, N.C. (1985), "Structural Similarity Between Legumin and Vicilin Storage Protein from Legumes",
EMBO Journal, Vol 5, 1111-1118.
Chu, S.S.C., Narayana, S. V. L., and Rosenstein, R. D. (1984), "1-Isopropyl-10-methylphenothiazine, C16H17NS.” Acta Cryst. C40, 1281-1283.|
Narayana, S. V. L., and Argos, P. (1984), "Residue Contacts in Protein Structures and Implications for Protein Folding", Int. J. Prot. and Peptides Res. 24, 25-39.
Zalkin, H., Argos, P., Narayana, S. V. L., Atiedeman, A. and Smith, J.M. (1984), "Identification of trypG-related Glutamin Amide Transfer Domain", J. Biol. Chem. 260, 3350-3354.
Narayana, S. V. L., Weininger, M.S., Heuss, K.L., and Argos, P. (1982), "A Method for Increasing Protein Crystal Lifetime During X-ray Exposure",J. Appl. Crystallogr. 15, 571-572.
Narayana, S. V. L. and Shrivastava, H.N. (1980), "Crystal and Molecular Structure of 2-sigma-bromo-sigma-tetrahydrosantonin”, J.C.S. Perkin Transaction II, 7, 1116-1118.
Krishnan, V., Datta, A., and Narayana, S. V. L. (1980), "Reaction of Selenium Sulfide with Triphenyl Compounds of Group V Elements",Inorganic Nucl. Chem. Letters 1, 517-522.
Narayana, S. V. L. and Shrivastava, H. N. (1980), "The Structure of Triphenylarsine Sulfide", Acta. Cryst. B37: 1186-1189.