Biplab Ghosh

Protein crystallography beamline (PX‐BL21) at Indus‐2 synchrotron

The protein crystallography beamline (PX-BL21), installed at the 1.5 T bending-magnet port at the Indian synchrotron (Indus-2), is now available to users. The beamline can be used for X-ray diffraction measurements on a single crystal of macromolecules such as proteins, nucleic acids and their complexes. PX-BL21 has a working energy range of 5-20 keV for accessing the absorption edges of heavy elements commonly used for phasing. A double-crystal monochromator [Si(111) and Si(220)] and a pair of rhodium-coated X-ray mirrors are used for beam monochromatization and manipulation, respectively. This beamline is equipped with a single-axis goniometer, Rayonix MX225 CCD detector, fluorescence detector, cryogenic sample cooler and automated sample changer. Additional user facilities include a workstation for on-site data processing and a biochemistry laboratory for sample preparation. In this article the beamline, other facilities and some recent scientific results are briefly described.

Crystallization and preliminary X-ray diffraction analysis of Xaa-Pro dipeptidase from Xanthomonas campestris

Xaa-Pro dipeptidase (XPD; prolidase; EC 3.4.13.9) specifically hydrolyzes dipeptides with a prolyl residue at the carboxy-terminus. Xanthomonas spp. possess two different isoforms of XPD (48 and 43 kDa) which share ∼24% sequence identity. The XPD of 43 kDa in size (XPD43) from Xanthomonas spp. is unusual as it lacks the strictly conserved tyrosine residue (equivalent to Tyr387 in Escherichia coli aminopeptidase P) that is suggested to be important in the proton-shuttle transfer required for catalysis in the M24B (MEROPS) family. Here, the crystallization and preliminary X-ray analysis of XPD43 from X. campestris (GenBank accession No. NP_637763) are reported. Recombinant XPD43 was crystallized using the microbatch-under-oil technique. Diffraction data were collected on the recently commissioned protein crystallography beamline (PX-BL21) at the Indian synchrotron (Indus-2, 2.5 GeV) to 1.83 Å resolution with 100% completeness. The crystal belonged to space group P212121, with unit-cell parameters a = 84.32, b = 105.51, c = 111.35 Å. Two monomers are expected to be present in the asymmetric unit of the crystal, corresponding to a solvent content of 58%. Structural analysis of XPD43 will provide new insights into the role of the conserved residues in catalysis in the M24B family.

Crystal structure and biochemical investigations reveal novel mode of substrate selectivity and illuminate substrate inhibition and allostericity in a subfamily of Xaa-Pro dipeptidases

Xaa-Pro dipeptidase (XPD) catalyzes hydrolysis of iminopeptide bond in dipeptides containing trans-proline as a second residue. XPDs are found in all living organisms and are believed to play an essential role in proline metabolism. Here, we report crystal structures and extensive enzymatic studies of XPD from Xanthomonas campestris (XPDxc), the first such comprehensive study of a bacterial XPD. We also report enzymatic activities of its ortholog from Mycobacterium tuberculosis (XPDmt). These enzymes are strictly dipeptidases with broad substrate specificities. They exhibit substrate inhibition and allostericity, as described earlier for XPD from Lactococcus lactis (XPDll). The structural, mutational and comparative data have revealed a novel mechanism of dipeptide selectivity and substrate binding in these enzymes. Moreover, we have identified conserved sequence motifs that distinguish these enzymes from other prolidases, thus defining a new subfamily. This study provides a suitable structural template for explaining unique properties of this XPDxc subfamily. In addition, we report unique structural features of XPDxc protein like an extended N-terminal tail region and absence of a conserved Tyr residue near the active site.

Gating role of His 72 in TmPurL enzyme uncovered by structural analyses and molecular dynamics simulations

Histidine is ubiquitous in enzyme active sites but its role is often difficult to ascribe due to ambiguity of protonation state and complex electrostatic and dynamic effects involved. In this study the role of His 72 in TmPurL, a glutamine amidotransferase (GAT) enzyme, is investigated. TmPurL is a large 66kDa enzyme that works as part of an even larger (>100kDa) multi-protein complex. This enzyme complex performs an essential step in the purine biosynthesis pathway by abstracting ammonia from a glutamine molecule and channeling it 30Å away into the active site of TmPurL, incorporating it into a purine biosynthesis intermediate. It is known that His 72 is important for substrate binding and possibly acts as a general base. Comparing apo and holo structural forms of this enzyme has revealed to us a possible gating function of His 72 that could regulate ammonia entry into the active site. Bimodal distribution of the χ1 dihedral angle of this amino acid in molecular dynamics simulations of 2μs supports the hypothesis. Different protonation states of His 72 were found to be conformationally distinct, providing a possible link between catalytic and gating roles of His 72. Ammonia channeling and allostery are discussed for GATs and more specifically for PurL family.

Structure of the human aminopeptidase XPNPEP3 and comparison of its in vitro activity with Icp55 orthologs: Insights into diverse cellular processes

The human aminopeptidase XPNPEP3 is associated with cystic kidney disease and TNF-TNFR2 cellular signaling. Its yeast and plant homolog Icp55 processes several imported mitochondrial matrix proteins leading to their stabilization. However, the molecular basis for the diverse roles of these enzymes in the cell is unknown. Here, we report the crystal structure of human XPNPEP3 with bound apstatin product at 1.65 Å resolution, and we compare its in vitro substrate specificity with those of fungal Icp55 enzymes. In contrast to the suggestions by earlier in vivo studies of mitochondrial processing, we found that these enzymes are genuine Xaa-Pro aminopeptidases, which hydrolyze peptides with proline at the second position (P1′). The mitochondrial processing activity involving cleavage of peptides lacking P1′ proline was also detected in the purified enzymes. A wide proline pocket as well as molecular complementarity and capping at the S1 substrate site of XPNPEP3 provide the necessary structural features for processing the mitochondrial substrates. However, this activity was found to be significantly lower as compared with Xaa-Pro aminopeptidase activity. Because of similar activity profiles of Icp55 and XPNPEP3, we propose that XPNPEP3 plays the same mitochondrial role in humans as Icp55 does in yeast. Both Xaa-Pro aminopeptidase and mitochondrial processing activities of XPNPEP3 have implications toward mitochondrial fitness and cystic kidney disease. Furthermore, the presence of both these activities in Icp55 elucidates the unexplained processing of the mitochondrial cysteine desulfurase Nfs1 in yeast. The enzymatic and structural analyses reported here provide a valuable molecular framework for understanding the diverse cellular roles of XPNPEP3.

Active site gate of M32 carboxypeptidases illuminated by crystal structure and molecular dynamics simulations

Enzyme gates are important dynamic features that regulate function. Study of these features is critical for understanding of enzyme mechanism. In this study, the active-site gate of M32 carboxypeptidases (M32CP) is illuminated. Only a handful of members of this family have been structurally and functionally characterized and various aspects of their activity and mechanism are yet not clarified. Here, crystal structure of putative M32CP from Deinococcus radiodurans (M32dr) was solved to 2.4Å resolution. Enzymatic assays confirmed its identity as a carboxypeptidase. Open and relatively closed conformations observed in the structure provided supporting evidence for previously hypothesized hinge motion in this family of enzymes. Molecular dynamics simulations of 1.5μs displayed distinct open and closed conformations revealing amplitude of the motion to be beyond what was observed in the crystal structure. Hinge region and anchoring region of this shell-type gate were identified. A small displacement of 3Å and a helical tilt of 9° propagated by the hinge region translates into a 10Å motion at the top of the gate. The dynamics of the gate was supported by our mutagenesis experiment involving formation of disulphide bond across helices of the gate. The nearly inactive mutant enzyme showed 65-fold increase in the enzymatic activity in presence of reducing agent. Further, while a previously proposed structural basis would have led to its classification in subfamily II, experimentally observed substrate length restriction places M32dr in subfamily I of M32CPs.

Crystal structure and dynamics of Spt16N-domain of FACT complex from Cicer arietinum.

The facilitates chromatin transcription (FACT) complex, a heterodimer of SSRP1 and Spt16 proteins, is an essential histone chaperone that transiently reorganizes nucleosomes during transcription, replication and repair. N-terminal domain of Spt16 subunit (Spt16N) is strictly conserved in all the known Spt16 orthologs. Genetic studies in yeast have revealed a partially redundant role of Spt16N for the FACT functionality. Here, we report the crystal structure of Spt16N from a plant origin (Spt16Nca, Cicer arietinum) and its comparisons with the known Spt16N structures from yeasts and human. The inter-domain angle in Spt16Nca is significantly different from that of the yeast and human Spt16N structures. Normal mode analysis and classical molecular dynamics simulations reveal inter-domain movement in Spt16Nca and later also shows conformational flexibility of the critical loops. Spt16Nca binds to histone H3/H4 complex, similar to its orthologs from yeast and human origins. Further, conservation of electrostatic surface potentials in Spt16N structures from evolutionary distinct domains of eukaryotes (plant, human and fungi) have provided the potential sites on Spt16N for histone interactions. The structural comparisons with M24 peptidases show that the hydrophobic pocket shielded by a flexible loop of C-terminal domain of Spt16N that may be functionally important.

Expression, purification, crystallization and preliminary X-ray diffraction analysis of acylpeptide hydrolase from Deinococcus radiodurans

Acylpeptide hydrolase (APH; EC 3.4.19.1), which belongs to the S9 family of serine peptidases (MEROPS), catalyzes the removal of an N-acylated amino acid from a blocked peptide. The role of this enzyme in mammalian cells has been suggested to be in the clearance of oxidatively damaged proteins as well as in the degradation of the β-amyloid peptides implicated in Alzheimers disease. Detailed structural information for the enzyme has been reported from two thermophilic archaea; both of the archaeal APHs share a similar monomeric structure. However, the mechanisms of substrate selectivity and active-site accessibility are totally different and are determined by inter-domain flexibility or the oligomeric structure. An APH homologue from a bacterium, Deinococcus radiodurans (APHdr), has been crystallized using microbatch-under-oil employing the random microseed matrix screening method. The protein crystallized in space group P21, with unit-cell parameters a = 77.6, b = 189.6, c = 120.4 Å, β = 108.4°. A Matthews coefficient of 2.89 Å(3) Da(-1) corresponds to four monomers, each with a molecular mass of ∼73 kDa, in the asymmetric unit. The APHdr structure will reveal the mechanisms of substrate selectivity and active-site accessibility in the bacterial enzyme. It will also be helpful in elucidating the functional role of this enzyme in D. radiodurans.

Crystallization and preliminary X-ray crystallographic analysis of an artificial molten-globular-like triosephosphate isomerase protein of mixed phylogenetic origin

A bioinformatics-based protein-engineering approach called consensus design led to the construction of a chimeric triosephosphate isomerase (TIM) protein called ccTIM (curated consensus TIM) which is as active as Saccharomyces cerevisiae TIM despite sharing only 58% sequence identity with it. The amino-acid sequence of this novel protein is as identical to native sequences from eukaryotes as to those from prokaryotes and shares some biophysical traits with a molten globular protein. Solving its crystal structure would help in understanding the physical implications of its bioinformatics-based sequence. In this report, the ccTIM protein was successfully crystallized using the microbatch-under-oil method and a full X-ray diffraction data set was collected to 2.2 Å resolution using a synchrotron-radiation source. The crystals belonged to space group C2221, with unit-cell parameters a=107.97, b=187.21, c=288.22 Å. Matthews coefficient calculations indicated the presence of six dimers in the asymmetric unit, with an approximate solvent content of 46.2%.

Crystal structure of a novel prolidase from Deinococcus radiodurans identifies new subfamily of bacterial prolidases

Xaa‐Pro peptidases (XPP) are dinuclear peptidases of MEROPS M24B family that hydrolyze Xaa‐Pro iminopeptide bond with a trans‐proline at the second position of the peptide substrate. XPPs specific towards dipeptides are called prolidases while those that prefer longer oligopeptides are called aminopeptidases P. Though XPPs are strictly conserved in bacterial and archaeal species, the structural and sequence features that distinguish between prolidases and aminopeptidases P are not always clear. Here, we report 1.4 Å resolution crystal structure of a novel XPP from Deinococcus radiodurans (XPPdr). XPPdr forms a novel dimeric structure via unique dimer stabilization loops of N‐terminal domains such that their C‐terminal domains are placed far apart from each other. This novel dimerization is also the consequence of a different orientation of N‐terminal domain in XPPdr monomer than those in other known prolidases. The enzymatic assays show that it is a prolidase with broad substrate specificity. Our structural, mutational, and molecular dynamics simulation analyses show that the conserved Arg46 of N‐terminal domain is important for the dipeptide selectivity. Our BLAST search found XPPdr orthologs with conserved sequence motifs which correspond to unique structural features of XPPdr, thus identify a new subfamily of bacterial prolidases.