Prof. M.R.N. Murthy Laboratory                 Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012, INDIA

Virus Crystallography

                Sesbania Mosaic Virus (SeMV) is an isometric, ss-RNA plant virus found infecting Sesbania grandiflora plants in fields near Tirupathi, South India. The three dimensional structure of SeMV was determined at 3.0 Å resolution. The icosahedral asymmetric unit was found to contain four ions (three calcium and an anion) and three protein subunits, designated A, B and C. The three calcium ions located at inter-subunit interfaces are related by quasi three-fold symmetry that relates the three independent subunits of the icosahedral asymmetric unit. The ligands to these ions emanate from two adjacent subunits and hence calcium plays the role of bonding the three subunits in the icosahedral asymmetric unit. The conformation of the C subunit is different from those of A and B in several segments of the polypeptide. In particular, the amino terminal 65 residues are disordered in the A and B subunits while only 38 residues are disordered in the C subunits. The additionally ordered part of the N-terminal arm of three C-subunits related by icosahedral 3-fold axis form a hydrogen bonded structure called the “b–annulus”. It has been suggested that this structure, also found in other viruses, is crucial for the error free assembly of the T=3 capsid. Structural studies on SeMV partially depleted of calcium suggest that one of the quasi-equivalent calcium ions is more easily displaced compared to the other two ions. Based on these studies, a plausible mechanism for the initiation of the disassembly of the virus has been suggested.

Crystal structure of the serine protease domain of Sesbania mosaic virus polyprotein

Crystal structures of Salmonella typhimurium biodegradative threonine deaminase (TdcB) and its complex with CMP provide structural insights into ligand induced    oligomerization and enzyme activation

Propionate kinase (TdcD) from Salmonella typhimurium

Structural studies on 2-methylisocitrate lyase (PrpB) involved in propionate metabolism in Salmonella typhimurium

    Propionate metabolism in Salmonella enterica serovar Typhimurium occurs via 2-methylcitric acid cycle. The last step of this cycle, the cleavage of 2-methylisocitrate to succinate and pyruvate is catalysed by 2-methylisocitrate lyase (EC 4.1.3.30). 2-Methylisocitrate lyase from Salmonella typhimurium corresponds to the PrpB protein of the prp operon involved in propionate oxidation. PrpB has been crystallized in forms suitable for structure determination by X-ray diffraction studies. X-ray crystal structure of the native and the pyruvate/Mg²⁺ bound PrpB from Salmonella typhimurium, has been determined at 2.1 and 2.3 Å, respectively. The structure closely resembles that of the E. coli enzyme. Unlike the E. coli PrpB, Mg²⁺ could not be located in the native Salmonella PrpB. Only in pyruvate bound PrpB structure, Mg²⁺ was found coordinated with pyruvate. Binding of pyruvate to PrpB seems to induce movement of the Mg²⁺  by 2.5 Å from its position found in E. coli native PrpB. In both the native enzyme and pyruvate/Mg²⁺ bound forms, the active site loop is completely disordered. Examination of the pocket in which pyruvate and glyoxalate bind to 2-methylisocitrate lyase and isocitrate lyase, respectively, reveals plausible rational for different substrate specifities of these two enzymes. Structural similarities in substrate and metal atom binding site as well as presence of similar residues in the active site suggest possible similarities in the reaction mechanism.Comparison of Salmonella typhimurium 2-methylisocitare lyase with the known bacterial isocitrate lyase reveals regions within the isocitrate lyase that are not present in PrpB, the smallest member of the family. These regions mainly comprise of amino and carboxy terminal ends as well as specific insertions within loops connecting β-strands and α-helices of the (αβ)₈ barrel. Sequence comparison among all known bacterial 2-methylisocitrate lyases and isocitrate lyases shows that Phe, Leu and Pro residues, which bind pyruvate in the case of 2-methylisocitrate lyase, and Trp, Phe and Thr, which bind glyoxalate in the case of isocitrate lyases, are strictly conserved, which seems to confer substrate specificity in these two enzymes. Similarities and differences in the structure of substrates, the active site region and the residues that interact with the ligands in these enzymes suggest similar reaction mechanisms in isocitrate lyases and 2-methylisocitrate lyases.

Diaminopropionate ammonia belongs to the family of PLP dependent enzymes and presumably is similar in structure to the b family (fold type II) of PLP dependent enzymes. Dap (Diaminopropionate) is the immediate precursor of the neurotoxins 3-oxalyl and 2,3 dioxalyl diaminopropionate, which cause neurolathyrism when ingested regularly or in large doses. These neurotoxins are present in Lathyrus sativus, a grain legume rich in proteins and capable of growing well in drought conditions. The presence of the neurotoxin precludes its exploitation as a major source of food. A study of the enzyme involved in the biosynthesis and degradation of the neurotoxin is a prerequisite for developing biotechnological methods for reducing its level in the seeds. DAP ammonia lyase from E.coli has been expressed, purified and crystallized and structural studies by X-ray diffraction have been initiated.

Structural studies on  Pf Adenylosuccinate Synthetase              

      Plasmodium falciparum malaria, with an annual morbidity of 300 million and mortality between 1 and 2 million is a growing worldwide health concern. Rapid emergence of drug resistance and absence of effective vaccines make the search for newer drug targets a continuous necessity. Metabolic pathways that are indispensable for parasite survival are an obvious choice as targets for the development of new antimalarials. The purine salvage pathway is one such potential target, as it provides the sole source of purine nucleotides for the parasite. Examination of the purine salvage pathway operative in the parasite indicates multiple avenues for inhibitor design. In addition to direct inhibitors of enzymes in the pathway, subversive substrates, which either inhibit down-stream enzymes or disrupt cellular processes by incorporation into the nucleotide pool, could also be effective drugs. Adenylosuccinate synthetase (AdSS) catalyzes the condensation of IMP with aspartate to form adenylosuccinate in the purine salvage pathway, in a reaction accompanied by the hydrolysis of GTP to GDP in the presence of Mg²⁺. The reaction proceeds in two steps, the first of which is the transfer of the g-phosphate of GTP to the O6 of IMP, to give 6–phosphoryl IMP. Aspartate then displaces the phosphate to give adenylosuccinate. This reaction is the first committed step in the synthesis of AMP from IMP, both in the de novo and salvage pathways for purine nucleotide synthesis. Regulation of this enzyme is involved in the maintenance of ATP/GTP ratios in the cell. Regulation is effected both by the products, GDP and adenylosuccinate, and, by the end products of the pathway, AMP and GMP. The P. falciparum genome is more than 70% A/T rich, thereby requiring maintenance of very different ATP/GTP ratios within the parasite as compared to the host. In this context, studies on the activity and regulation of this parasite enzyme gain importance. The cloning, recombinant expression, purification and preliminary kinetic characterization of PfAdSS have been carried out in Prof. Hemalatha Balaram’s laboratory at the Jawaharlal Nehru Institute for Advanced Scientific Research, Bangalore. It was previously reported that the parasite enzyme, like its homologs from other species, is active as a homodimer, with K_m values for IMP and GTP similar to the reported values for the enzyme from other sources. However, the K_m for aspartate is close to 1.5 mM, about 5 fold higher than that seen with the E. coli enzyme and the mouse basic isozyme for which three dimensional structures are available. This value is in fact comparable to that of the acidic mouse non-muscle isozyme, which has an aspartate K_m of 1.0 mM.  Binding behaviour of PfAdSS on ion exchange columns indicates that it is an acidic enzyme. We have determined the crystal structure of adenylosuccinate synthetase from the malaria parasite, Plasmodium falciparum, complexed to 6-phosphoryl IMP, GDP, Mg²⁺ and the aspartate analogue, hadacidin at 2Å resolution. The structure of the fully ligated PfAdSS is largely identical to the fully ligated structures reported for the mouse and E. coli enzymes. The dimer interface of PfAdSS is different with a pronounced excess of positively charged residues. These differences provide a basis for the design of species specific inhibitors of the enzyme. The differences observed in the kinetic behaviour of PfAdSS can be attributed to small structural variations seen in the GTP binding pocket. Residues in the Switch loop, which respond to IMP binding, have additional hydrogen bonding interactions with the ribose hydroxyls of GDP and also, with residues in the GTP loop. GDP also makes additional hydrogen bonds with Thr307 in the aspartate loop. These interactions may account for the ordered binding of substrates that is observed in PfAdSS. However, in the absence of unligated and partially ligated structures of PfAdSS, these inferences drawn from the available E. coli and mouse structures remain conjectural. The kinetic and structural data highlight the difficulty in extrapolating from structure to mechanism with subtle structural differences having significant mechanistic implications. As only the salvage pathway is operative in P. falciparum, AdSS represents an important drug target. It is interesting to note that allopurinol ribonucleoside phosphate (ARP) is a substrate for Leishmania donovonii AdSS (LdAdSS) and only a weak inhibitor of the mammalian basic isozyme. Succinylation of ARP by LdAdSS and subsequent incorporation into the parasite’s nucleotide pool is responsible for the selective toxicity of this IMP analogue to the parasite.  The structure of P. falciparum AdSS opens up the possibility of exploiting differences in protein-ligand interactions between the parasite and host enzyme for species-specific inhibitor design.

Structural studies on Plasmodium falciparum triosephosphate isomerase

Triosephosphate isomerase (TIM) catalysis the isomerization between dihydroxyacetone phosphate and glyceraldehyde-3-phosphate. The structure and catalysis of this housekeeping enzyme has been extensively investigated from several sources as an ideal model for the investigation of structure-function relationship of protein enzymes. Glycolytic enzymes of the malarial parasite Plasmodium falciparum (Pf) have also been studied as potential targets for antimalarial drug design. The structure of Pf-TIM was determined at 2.2 Å in my laboratory. TIM structure consists of an 8-stranded, parallel b-barrel with the loops at the carboxyl end of the barrel contributing residues important for catalysis. This classical “TIM-barrel’ structure is also found in a large number of other proteins unrelated to TIM in sequence or function. Comparison of the Pf-TIM structure to that of the human enzyme provided information on potential sites that can be exploited for the design of inhibitor molecules specific to the parasite enzyme. It was found that residue 96, which is phenylalanine in the plasmodium enzyme and serine in other TIMs affects the dynamics of the catalytic loop (loop6). The movement of this loop is intimately connected with the function of the enzyme. Therefore, the presence of phenylalanine at 96 might be useful in developing lead compounds against malarial infections. With a view of obtaining more information on the interactions responsible for inhibitor binding, structures of complexes of Pf-TIM with a variety of inhibitors were determined. (3-phosphoglycerate,3PG; Glycerol-3-phosphate, G3P; 2-phosphoglycerate, 2PG; phosphoglycolate, PG). Loop 6 (catalytic loop) is in an “open” conformation in Pf-TIM even when G3P and 3PG are bound, in contrast to earlier observations in other known TIM structures where this loop undergoes large conformational changes upon inhibitor binding to a “closed conformation”. Detailed analysis suggests that the residue 96, which is close to the active site and is a Phe in the parasite enzyme, in contrast to Ser in most other organisms, is responsible for the lack of closure of loop 6 upon inhibitor binding. These differences could of importance in molecular structure based drug design against malarial infections. In two different crystal forms of Plasmodium falciparum TIM -phosphoglycolate complex, the catalytic loops are found in open and closed conformations, respectively. In the crystal structure of Plasmodium falciparum TIM complexed to the inhibitor 2-phosphoglycerate (2PG) determined at 1.1Å resolution, the active site loop in one of the subunits is in both “open” and “closed” conformations, although the “open” form is predominant. These studies have provided deeper insights into the relationship of different residues at the active site to the dynamics of the catalytic loop and enzyme function.

 Protein dynamics by X-ray diffraction

                Atomic displacement parameters (ADPs or B-values) obtained from X-ray refinement of proteins at high resolution, when expressed in units of standard deviation about the their mean value (B’- factor) at the Ca atoms were shown to have a characteristic frequency distributions. The distribution fits the superposition of two Gaussian functions very well. It was shown that these distributions provide information on the relationship between amino acid residues and the rigidity imposed by them on the polypeptide chain.   Examination of the correlation coefficients (CCs) between the mean B-values of main chain and side chain atoms in selected high-resolution protein structures shows dependence on the package used for refinement (X-PLOR, PROLSQ or TNT). It is likely that these differences are related to the different refinement protocols or weighting schemes followed by investigators and suggested the necessity of improvement in the constraints used in protein refinement. Analysis of the ADPs obtained from mesophiles and thermophiles has suggested a potential relationship between protein stability and dynamics. It was found that Ser and Thr have lesser flexibility in thermophiles than in mesophiles. In addition, composition of Glu and Lys in high B-value regions of thermophiles is higher and that of Ser and Thr is lower. Comparative analysis of the ADPs in homologous proteins shows that the flexible and rigid regions in the three-dimensional fold of proteins remain largely conserved during the course of evolution, reflecting the importance of dynamics in protein structure or function. Analysis of the relationship between the flexibility of the protein molecule and its conformation shows that the flexibility of different segments of the polypeptide is sensitive to the conformation of the side chains as well as that of the main chain.

 Studies on calcium binding EF hand proteins

Thermodynamics of calcium binding to the 4 sites present in a calmodulin (CAM) homologue, a calcium binding protein (CaBP) from Entamoeba histolytica has been investigated in detail. The residues important for calcium binding have been probed by site-specific mutations followed by spectroscopic studies.  The modulation of calcium binding by three external parameters-pH, ligand co-ordinator EGTA and fragmentor voltage was investigated by mass spectrometry. Calcium binding follows expected patterns at highly acidic and alkaline pH with the preponderance of the apo and the completely saturated forms, respectively. Surprisingly, additional non-specific binding was observed near neutral pH., suggesting non-specific sites other than the EF-hands for calcium binding. Studies on EGTA chelation and effects of fragmentor voltage in mass spectroscopy showed co-operativity in calcium removal in at least one of the domains. Similar studies on a smaller construct containing the two high affinity carboxy terminal sites have been performed. Earlier analyses of the amino acid sequences of calmodulins have provided clear evidence for a characteristic double gene duplication that has occurred early in the evolutionary history of this family of proteins. These analyses have been extended to the three dimensional structures and the biophysical properties of the sequence segments of EF-hands. The evolutionary history that clearly shows up in sequences is largely masked in the conformation of individual EF-hands. Some evidence for the proposed gene duplication is, however, implicit in the apo-holo structural transitions of the EF-hands. The profile of amino acid properties that might be significant for calcium binding clearly reflects the gene duplication. These profiles also provide insightful information on the calcium affinity of the EF-hand motifs and the nature of amino acid residues that constitute them.

Studies on mutants of thymidylate synthase

                Structures for two crystal forms of an R178F mutant of L. casei thymidylate synthase with c edges of 230.4 and 244 were determined. Comparison of these structures, and these to the other structures with intermediate cell parameters reported in the literature indicates that there are no large changes in the dimeric structure of TS in these forms. Although there is a large change in the unit cell volume, the molecular contacts in the crystal structures are nearly invariant.  The transformation appears to result from concerted small changes in the molecular structure of TS and in inter-dimer contacts in the crystal structure. These observations corroborate the general impression that protein structures are not drastically altered by substitution mutations and the changes in sequence lead to minor alterations in the backbone fold and tertiary packing interactions.

Studies on the evolution of plant seed inhibitors

                Plant seed inhibitors provide an excellent opportunity to understand aspects protein evolution. Using the sequences of plant amylase / chymotrypsin double headed inhibitors available in the SWISS-PROT database, it was shown that the phylogeny of these sequences reflect their oligomeric structure and enzyme specificity rather than the botanical class of the host plant. Thus the functional differentiation of these inhibitors occurred early in evolution. In contrast, analysis of the Bowman-Birk inhibitor sequences suggests that the inhibitors belonging to monocots and dicots form distinct families. Further division of monocot and dicot inhibitors is on the basis of enzyme specificities. Thus, the two classes of inhibitors are good examples of contrasting features of molecular evolution. The ability of Bowman-Birk inhibitors belonging to different classes (say Kunitz and Squash) to inhibit the same enzyme implies convergent evolution. Analysis of the active site sequences of various plant inhibitors revealed that the 12-residue segment responsible for inhibition displays a phylogeny similar to those obtained from full sequences.

   Structural studies on polyamine interactions

Structures of polyamines complexed with several other ubiquitous biomolecules were determined. Systematic analysis of these structures has shown that the specificity of polyamine function arises due to the multiplicity of interactions provided by them. These studies have revealed the relative strengths of hydrogen bonding, electrostatic and Vander Waals interactions of these molecules.  Extending these studies, the possible reasons for polyamines binding only to certain classes of viral genomes were investigated. All published plant viral genomes were analyzed and it was shown that the viruses that bind polyamines are characterized by a highly significant biased distribution of different types of oligonucleotides.  This skew-ness results in the possibility of a large number of short stretches of double helical regions in the nucleic acid of viral genomes binding polyamines.  

 Protein Folding

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