Karthe Ponnuraj

Crystal structure of the first plant urease from jack bean: 83 years of journey from its first crystal to molecular structure.

Urease, a nickel-dependent metalloenzyme, is synthesized by plants, some bacteria, and fungi. It catalyzes the hydrolysis of urea into ammonia and carbon dioxide. Although the amino acid sequences of plant and bacterial ureases are closely related, some biological activities differ significantly. Plant ureases but not bacterial ureases possess insecticidal properties independent of its ureolytic activity. To date, the structural information is available only for bacterial ureases although the jack bean urease (Canavalia ensiformis; JBU), the best-studied plant urease, was the first enzyme to be crystallized in 1926. To better understand the biological properties of plant ureases including the mechanism of insecticidal activity, we initiated the structural studies on some of them. Here, we report the crystal structure of JBU, the first plant urease structure, at 2.05 A resolution. The active-site architecture of JBU is similar to that of bacterial ureases containing a bi-nickel center. JBU has a bound phosphate and covalently modified residue (Cys592) by beta-mercaptoethanol at its active site, and the concomitant binding of multiple inhibitors (phosphate and beta-mercaptoethanol) is not observed so far in bacterial ureases. By correlating the structural information of JBU with the available biophysical and biochemical data on insecticidal properties of plant ureases, we hypothesize that the amphipathic beta-hairpin located in the entomotoxic peptide region of plant ureases might form a membrane insertion beta-barrel as found in beta-pore-forming toxins.

Evidence for the "dock, lock, and latch" ligand binding mechanism of the staphylococcal microbial surface component recognizing adhesive matrix molecules (MSCRAMM) SdrG.

Staphylococcus epidermidis is an opportunistic pathogen and a major cause of foreign body infections. The S. epidermidis fibrinogen (Fg)-binding adhesin SdrG is necessary and sufficient for the attachment of this pathogen to Fg-coated materials. Based largely on structural analyses of the ligand binding domain of SdrG as an apo-protein and in complex with a Fg-like peptide, we proposed that SdrG follows a “dock, lock, and latch” mechanism to bind to Fg. This binding mechanism involves the docking of the ligand in a pocket formed between two SdrG subdomains followed by the movement of a C-terminal extension of one subdomain to cover the ligand and to insert and complement a β-sheet in a neighboring subdomain. These proposed events result in a greatly stabilized closed conformation of the MSCRAMM-ligand complex. In this report, we describe a biochemical analysis of the proposed conformational changes that SdrG undergoes upon binding to its ligand. We have introduced disulfide bonds into SdrG to stabilize the open and closed forms of the apo-form of the MSCRAMM. We show that the stabilized closed form does not bind to the ligand and that binding can be restored in the presence of reducing agents such as dithiothreitol. We have also used Förster resonance energy transfer to dynamically show the conformational changes of SdrG upon binding to its ligand. Finally, we have used isothermic calorimetry to determine that hydrophobic interactions between the ligand and the protein are responsible for re-directing the C-terminal extension of the second subdomain required for triggering the β-strand complementation event.

The Enterococcus faecalis MSCRAMM ACE Binds Its Ligand by the Collagen Hug Model

We have determined the crystal structure of the ligand binding segment of the Enterococcus faecalis collagen binding MSCRAMM ACE (microbial surface components recognizing adhesive matrix molecules adhesin of collagen from enterococci). 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 interface 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. Replacing residues in the putative collagen binding trench of ACE N2 with Ala residues affected collagen binding. A closed conformation of ACE stabilized by an engineered disulfide bond is 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.

Structural and functional studies on urease from pigeon pea (Cajanus cajan)

Urease is an enzyme that catalyzes the hydrolysis of urea, forming ammonia and carbon dioxide, and is found in plants, microorganisms and invertebrates. Although plant and bacterial ureases are closely related at amino acid and at the structural level, the insecticidal activity is seen only in the plant ureases. In contrast, both plant and bacterial ureases exhibit antifungal activity. These two biological properties are independent of its ureolytic activity. However, till date the mechanism(s) behind the insecticidal and fungicidal activity of ureases are not clearly understood. Here we report the crystal structure of pigeon pea urease (PPU, Cajanus cajan) which is the second structure from the plant source. We have deduced the amino acid sequence of PPU and also report here studies on its stability, insecticidal and antifungal activity. PPU exhibits cellulase activity. Based on the structural analysis of PPU and docking studies with cellopentoase we propose a possible mechanism of antifungal activity of urease.

Crystal structure of glutamine receptor protein from Sulfolobus tokodaii strain 7 in complex with its effector l-glutamine: implications of effector binding in molecular association and DNA binding

Genome analyses have revealed that members of the Lrp/AsnC family of transcriptional regulators are widely distributed among prokaryotes, including both bacteria and archaea. These regulatory proteins are involved in cellular metabolism in both global and specific manners, depending on the availability of the exogenous amino acid effectors. Here we report the first crystal structure of glutamine receptor protein (Grp) from Sulfolobus tokodaii strain 7, in the ligand-free and glutamine-bound (Grp-Gln) forms. Although the overall structures of both molecules are similar, a significant conformational change was observed at the ligand [l-glutamine (Gln)] binding site in the effector domain, which may be essential for further stabilization of the octameric structure, and in turn for facilitating DNA binding. In addition, we predicted promoter for the grp gene, and these analyses suggested the importance of cooperative binding to the protein. To gain insights into the ligand-induced conformational changes, we mutated all of the ligand-binding residues in Grp, and revealed the importance of Gln binding by biochemical and structural analyses. Further structural analyses showed that Y77 is crucial for ligand binding, and that the residues T132 and T134, which are highly conserved among the Lrp family of proteins, fluctuates between the active and inactive conformations, thus affecting protein oligomerization for DNA binding.

Structure of laminin-binding adhesin (Lmb) from Streptococcus agalactiae.

Adhesion/invasion of pathogenic bacteria is a critical step in infection and is mediated by surface-exposed proteins termed adhesins. The crystal structure of recombinant Lmb, a laminin-binding adhesin from Streptococcus agalactiae, has been determined at 2.5 A resolution. Based on sequence and structural homology, Lmb was placed into the cluster 9 family of the ABC (ATP-binding cassette) transport system. The structural organization of Lmb closely resembles that of ABC-type solute-binding proteins (SBPs), in which two structurally related globular domains interact with each other to form a metal-binding cavity at the interface. The bound zinc in Lmb is tetrahedrally coordinated by three histidines and a glutamate from both domains. A comparison of Lmb with other metal transporters revealed an interesting feature of the dimerization of molecules in the crystallographic asymmetric unit in all zinc-binding transporters. A closer comparison of Lmb with the zinc-binding ZnuA from Escherichia coli and Synechocystis 6803 suggested that Lmb might undergo a unique structural rearrangement upon metal binding and release. The crystal structure of Lmb provides an impetus for further investigations into the molecular basis of laminin binding by human pathogens. Being ubiquitous in all serotypes of group B streptococcus (GBS), the structure of Lmb may direct the development of an efficient vaccine.

The Crystal Structure of Cobra Venom Factor, a Cofactor for C3- and C5-Convertase CVFBb

Cobra venom factor (CVF) is a functional analog of human complement component C3b, the active fragment of C3. Similar to C3b, in human and mammalian serum, CVF binds factor B, which is then cleaved by factor D, giving rise to the CVFBb complex that targets the same scissile bond in C3 as the authentic complement convertases C4bC2a and C3bBb. Unlike the latter, CVFBb is a stable complex and an efficient C5 convertase. We solved the crystal structure of CVF, isolated from Naja naja kouthia venom, at 2.6 A resolution. The CVF crystal structure, an intermediate between C3b and C3c, lacks the TED domain and has the CUB domain in an identical position to that seen in C3b. The similarly positioned CUB and slightly displaced C345c domains of CVF could play a vital role in the formation of C3 convertases by providing important primary binding sites for factor B.

Cloning, expression, purification and ligand binding studies of novel fibrinogen-binding protein FbsB of Streptococcus agalactiae

Fibrinogen (Fg) is often a common site for bacterial recognition. In Streptococcus agalactiae, two surface proteins that recognize Fg are FbsA and FbsB. FbsA and the N-terminal region of FbsB have been shown to bind to human Fg, while the C-terminal region of FbsB [FbsB(C)] has been speculated to bind to bovine Fg. This C-terminal region which is conserved in many of the S. agalactiae strains was tested for binding to bovine Fg. For this, FbsB(C) was cloned, expressed and purified. Dot blot, Western blot and ELISA experiments carried out with the purified protein showed that FbsB(C) has the ability to bind to bovine Fg. It was also observed that other than binding to the native form of Fg, FbsB(C) also has the ability to bind to the Fg subunits when reduced. On studying the influence of Ca(2+) on the FbsB(C)-bovine Fg binding it was observed that the addition of Ca(2+) in the assay experiment greatly stimulated the binding. When the primary structure of FbsB(C) was analyzed, it was seen that other than similarities with strains of the same organism, it does not have any similarity with any protein characterized so far. In addition to this, its secondary structure component analysis by circular dichroism revealed that it is composed mainly of alpha helices and random coils unlike other Fg-binding surface proteins where beta sheets are dominant. FbsB(C) indeed is a novel protein and understanding the mechanism of its interaction with Fg would be useful in developing strategies to fight against infections by Streptococcus.

Crystal structure of ACE19, the collagen binding subdomain of Enterococus faecalis surface protein ACE

Enterococus faecalis is an extracellular pathogen known to cause many clinical infections, such as septicemia, bacteremia, and urinary tract infections.1 The bacterial pathogenesis is a complex process at the molecular level and the bacterial adherence to extracellular matrix components like collagen, fibrinogen, and fibronectin is a prerequisite for pathogenesis. Gram-positive bacteria are endowed with adhesive proteins termed as MSCRAMMs (Microbial Surface Component Recognizing Adhesive Matrix Molecules), located on the surface of the microbe to mediate such attachment. Characterization of several MSCRAMMs from various bacterial species revealed that they are structurally similar.2 In general, they contain an N-terminal signal peptide followed by (i) a ligand binding A region that is composed of two or more subdomains, each adopting a novel immunoglobulin-like (DEv-IgG) fold3; (ii) a B region that is made up of short repeat sequences; and (iii) the C-terminal end that contains the cell wall anchoring LPXTG motif, a hydrophobic transmembrane region (M), and a positively charged cytoplasmic tail (c), features that are essential for sorting these proteins to the cell wall.2 ACE is a collagen binding MSCRAMM of E. faecalis.4,5 The structural characteristics of ACE are similar to that of collagen binding protein CNA of Staphylococcus aureus [Fig. 1(A)], which has been studied in significant detail both in structural and biochemical aspects.6–10 The 55kDa ligand binding A region of CNA (termed CNA55) contains three subdomains namely N1, N2, and N3 and the minimum collagen binding region was localized to a 19-kDa N2 subdomain (termed CNA19) and its crystal structure was solved earlier in our laboratory.8 Recently, we determined the crystal structure of N1N2 subdomain of CNA (termed CNA35) both as an apo-protein and in complex with a synthetic collagen triple helix peptide. On the basis of these two crystal structures, we proposed a ‘‘Collagen Hug’’ binding mechanism for the association of CNA with collagen.11 The crystal structure of CNA35collagen complex revealed that collagen-like triple helical peptide mainly interacts with residues present in the ‘‘trench’’ region of the N2 domain, however, N1 domain also contributes critical residues for the stabilization of complex by sequestering critical regions of the ligand, explaining the 10-fold higher ligand binding affinity of CNA35 (N1 and N2) compared with CNA19. The 40-kDa A-region of ACE (termed ACE40) was predicted to have two subdomains namely N1 and N2. CNA55 and ACE40 share significant sequence similarity and particularly the minimum ligand binding region of CNA (CNA19) exhibits high degree of similarity (about 95%) with the corresponding region of ACE40 (i.e. ACE19). Previous modeling and spectroscopic studies predicted that the backbone folding of ACE19 is highly

Crystallization and preliminary X-ray crystallographic analysis of Ace: a Collagen-binding MSCRAMM from Enterococcus faecalis

Ace is a collagen-binding bacterial cell surface adhesin from Enterococcus faecalis. The collagen-binding domain of Ace (termed Ace40) and its truncated form Ace19 have been crystallized by the vapor-diffusion hanging-drop method. Ace19 was crystallized in two different crystal forms. A complete 1.65 A data set has been collected on the orthorhombic crystal form with unit cell parameters a=38.43 b=48.91 and c=83.73 A. Ace40 was crystallized in the trigonal space group P3(1)21 or P3(2)21 with unit cell parameters a=b=80.24, c=105.91 A; alpha=beta=90 and gamma=120 degrees. A full set of X-ray diffraction data was collected to 2.5 A. Three heavy atom derivative data sets have been successfully obtained for Ace19 crystals and structural analysis is in progress.