Amit Bhardwaj

Crystal structures of native and xylosaccharide-bound alkali thermostable xylanase from an alkalophilic Bacillus sp. NG-27: structural insights into alkalophilicity and implications for adaptation to polyextreme conditions.

Crystal structures are known for several glycosyl hydrolase family 10 (GH10) xylanases. However, none of them is from an alkalophilic organism that can grow in alkaline conditions. We have determined the crystal structures at 2.2 Å of a GH10 extracellular endoxylanase (BSX) from an alkalophilic Bacillus sp. NG‐27, for the native and the complex enzyme with xylosaccharides. The industrially important enzyme is optimally active and stable at 343 K and at a pH of 8.4. Comparison of the structure of BSX with those of other thermostable GH10 xylanases optimally active at acidic or close to neutral pH showed that the solvent‐exposed acidic amino acids, Asp and Glu, are markedly enhanced in BSX, while solvent‐exposed Asn was noticeably depleted. The BSX crystal structure when compared with putative three‐dimensional homology models of other extracellular alkalophilic GH10 xylanases from alkalophilic organisms suggests that a protein surface rich in acidic residues may be an important feature common to these alkali thermostable enzymes. A comparison of the surface features of BSX and of halophilic proteins allowed us to predict the activity of BSX at high salt concentrations, which we verified through experiments. This offered us important lessons in the polyextremophilicity of proteins, where understanding the structural features of a protein stable in one set of extreme conditions provided clues about the activity of the protein in other extreme conditions. The work brings to the fore the role of the nature and composition of solvent‐exposed residues in the adaptation of enzymes to polyextreme conditions, as in BSX.

Characterizing TDP-43 interaction with its RNA targets

One of the most important functional features of nuclear factor TDP-43 is its ability to bind UG-repeats with high efficiency. Several cross-linking and immunoprecipitation (CLIP) and RNA immunoprecipitation-sequencing (RIP-seq) analyses have indicated that TDP-43 in vivo can also specifically bind loosely conserved UG/GU-rich repeats interspersed by other nucleotides. These sequences are predominantly localized within long introns and in the 3′UTR of various genes. Most importantly, some of these sequences have been found to exist in the 3′UTR region of TDP-43 itself. In the TDP-43 3′UTR context, the presence of these UG-like sequences is essential for TDP-43 to autoregulate its own levels through a negative feedback loop. In this work, we have compared the binding of TDP-43 with these types of sequences as opposed to perfect UG-stretches. We show that the binding affinity to the UG-like sequences has a dissociation constant (Kd) of ∼110 nM compared with a Kd of 8 nM for straight UGs, and have mapped the region of contact between protein and RNA. In addition, our results indicate that the local concentration of UG dinucleotides in the CLIP sequences is one of the major factors influencing the interaction of these RNA sequences with TDP-43.

Genome-wide transcriptome and proteome analyses of tobacco psaA and psbA deletion mutants

Photosynthesis in higher land plants is a complex process involving several proteins encoded by both nuclear and chloroplast genomes that require a highly coordinated gene expression. Significant changes in plastid differentiation and biochemical processes are associated with the deletion of chloroplast genes. In this study we report the genome-wide responses caused by the deletion of tobacco psaA and psbA genes coding core components of photosystem I (PSI) and photosystem II (PSII), respectively, generated through a chloroplast genetic engineering approach. Transcriptomic and quantitative proteomic analysis showed the down regulation of specific groups of nuclear and chloroplast genes involved in photosynthesis, energy metabolism and chloroplast biogenesis. Moreover, our data show simultaneous activation of several defense and stress responsive genes including those involved in reactive oxygen species (ROS) scavenging mechanisms. A major finding is the differential transcription of the plastome of deletion mutants: genes known to be transcribed by the plastid encoded polymerase (PEP) were generally down regulated while those transcribed by the nuclear encoded polymerase (NEP) were up regulated, indicating simultaneous activation of multiple signaling pathways in response to disruption of PSI and PSII complexes. The genome wide transcriptomic and proteomic analysis of the ∆psaA and ∆psbA deletion mutants revealed a simultaneous up and down regulation of the specific groups of genes located in nucleus and chloroplasts suggesting a complex circuitry involving both retrograde and anterograde signaling mechanisms responsible for the coordinated expression of nuclear and chloroplast genomes.

Emerging role of N- and C-terminal interactions in stabilizing (β/α)8 fold with special emphasis on Family 10 xylanases

Xylanases belong to an important class of industrial enzymes. Various xylanases have been purified and characterized from a plethora of organisms including bacteria, marine algae, plants, protozoans, insects, snails and crustaceans. Depending on the source, the enzymatic activity of xylanases varies considerably under various physico-chemical conditions such as temperature, pH, high salt and in the presence of proteases. Family 10 or glycosyl hydrolase 10 (GH10) xylanases are one of the well characterized and thoroughly studied classes of industrial enzymes. The TIM-barrel fold structure which is ubiquitous in nature is one of the characteristics of family 10 xylanases. Family 10 xylanases have been used as a “model system” due to their TIM-barrel fold to dissect and understand protein stability under various conditions. A better understanding of structure-stability-function relationships of family 10 xylanases allows one to apply these governing molecular rules to engineer other TIM-barrel fold proteins to improve their stability and retain function(s) under adverse conditions. In this review, we discuss the implications of N-and C-terminal interactions, observed in family 10 xylanases on protein stability under extreme conditions. The role of metal binding and aromatic clusters in protein stability is also discussed. Studying and understanding family 10 xylanase structure and function, can contribute to our protein engineering knowledge.

Structural and biochemical characterization of an RNA/DNA binding motif in the N-terminal domain of RecQ4 helicases

The RecQ4 helicase belongs to the ubiquitous RecQ family but its exact role in the cell is not completely understood. In addition to the helicase domain, RecQ4 has a unique N-terminal part that is essential for viability and is constituted by a region homologous to the yeast Sld2 replication initiation factor, followed by a cysteine-rich region, predicted to fold as a Zn knuckle. We carried out a structural and biochemical analysis of both the human and Xenopus laevis RecQ4 cysteine-rich regions, and showed by NMR spectroscopy that the Xenopus fragment indeed assumes the canonical Zn knuckle fold, whereas the human sequence remains unstructured, consistent with the mutation of one of the Zn ligands. Both the human and Xenopus Zn knuckles bind to a variety of nucleic acid substrates, with a mild preference for RNA. We also investigated the effect of a segment located upstream the Zn knuckle that is highly conserved and rich in positively charged and aromatic residues, partially overlapping with the C-terminus of the Sld2-like domain. In both the human and Xenopus proteins, the presence of this region strongly enhances binding to nucleic acids. These results reveal novel possible roles of RecQ4 in DNA replication and genome stability.

Crystallization and preliminary X-ray study of a family 10 alkali-thermostable xylanase from alkalophilic Bacillus sp. strain NG-27

Xylanases (EC 3.2.1.8) catalyze the hydrolysis of beta-1,4-glycosidic linkages within xylan, a major hemicellulose component in the biosphere. The extracellular endoxylanase (XylnA) from the alkalophilic Bacillus sp. strain NG-27 belongs to family 10 of the glycoside hydrolases. It is active at 343 K and pH 8.4. Moreover, it has attractive features from the point of view of utilization in the paper pulp, animal feed and baking industries since it is an alkali-thermostable protein. In this study, XylnA was purified from the native host source and crystallized by the hanging-drop vapour-diffusion method. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 174.5, b = 54.7, c = 131.5 A, beta = 131.2 degrees, and diffract to better than 2.2 A resolution.

REVISITING RUBISCO AS A PROTEIN SUBSTRATE FOR INSECT MIDGUT PROTEASES

Gene fragments encoding the large subunit (LS) of Rubisco (RBCL) were cloned from various species of host plants of phytophagous Lepidoptera and expressed as recombinant proteins in Escherichia coli. Recombinant RBCLs were compared among each other along with casein and native Rubisco as proteinaceous substrates for measuring total midgut protease activities of fourth instar larvae of Helicoverpa armigera feeding on casein, Pieris brassicae feeding on cauliflower, and Antheraea assamensis feeding on Litsea monopetala and Persea bombycina. Cognate rRBCL (from the pertinent host plant species) substrates performed similar to noncognate rRBCL reflecting the conserved nature of encoding genes and the versatile use of these recombinant proteins. Casein and recombinant RBCL generally outperformed native Rubisco as substrates, except where inclusion of a reducing agent in the enzyme assay likely unfolded the plant proteins. Levels of total midgut protease activities detected in A. assamensis larvae feeding on two primary host species were similar, suggesting that the suite(s) of digestive enzymes in these insects could hydrolyze a plant protein efficiently. Protease activities detected in the presence of protease inhibitors and the reducing agent dithiothreitol (DTT) suggested that recombinant RBCL was a suitable protein substrate for studying insect proteases using in vitro enzyme assays and substrate zymography.

Structural insights into N‐terminal to C‐terminal interactions and implications for thermostability of a (β/α)8‐triosephosphate isomerase barrel enzyme

Although several factors have been suggested to contribute to thermostability, the stabilization strategies used by proteins are still enigmatic. Studies on a recombinant xylanase from Bacilllus sp. NG‐27 (RBSX), which has the ubiquitous (β/α)8‐triosephosphate isomerase barrel fold, showed that just a single mutation, V1L, although not located in any secondary structural element, markedly enhanced the stability from 70 °C to 75 °C without loss of catalytic activity. Conversely, the V1A mutation at the same position decreased the stability of the enzyme from 70 °C to 68 °C. To gain structural insights into how a single extreme N‐terminus mutation can markedly influence the thermostability of the enzyme, we determined the crystal structure of RBSX and the two mutants. On the basis of computational analysis of their crystal structures, including residue interaction networks, we established a link between N‐terminal to C‐terminal contacts and RBSX thermostability. Our study reveals that augmenting N‐terminal to C‐terminal noncovalent interactions is associated with enhancement of the stability of the enzyme. In addition, we discuss several lines of evidence supporting a connection between N‐terminal to C‐terminal noncovalent interactions and protein stability in different proteins. We propose that the strategy of mutations at the termini could be exploited with a view to modulate stability without compromising enzymatic activity, or in general, protein function in diverse folds where N and C termini are in close proximity.

Role of long-range aromatic clique and community in protein stability.

Aromatic interactions make an important contribution to protein structure, function, folding and have attracted intense study. Earlier studies on a recombinant xylanase from Bacillus sp. NG-27 (RBSX), which has the ubiquitous (beta/alpha)8-triosephosphate isomerase barrel fold showed that three aromatic residues to alanine substitutions, in the N-terminal and C-terminal regions, significantly decreased the stability of the enzyme. Of these mutations, F4A mutation decreased the stability of the enzyme by ∼4 degree C, whereas W6A mutation and Y343A mutation remarkably decreased the stability of the enzyme by ∼10 degree C. On the other hand, the F4W mutation did not affect the thermal stability of RBSX. We provide here a network perspective of aromatic-aromatic interactions in terms of aromatic clique community and long-range association. Our study reveals that disruption of long-range k-clique aromatic interaction cluster holding the N- and C-terminal regions are associated with the decreased stability of the enzyme. The present work reiterates as well as expands on those findings concerning the role of interactions between the N- and C-terminus in protein stability. Furthermore, comparative analyses of crystal structures of homologous pairs of proteins from thermophilic and mesophilic organisms emphasize the prevalence of long-range k-clique communities of aromatic interaction that may be playing an important role and highlights an additional source of stability in thermophilic proteins. The design principle based on clustering of long-range aromatic residues in the form of aromatic-clique and clique community may be effectively applied to enhance the stability of enzymes for biotechnological applications. Database The coordinates o fF4A, F4W, W6A, and Y343A are deposited in the PDB database under the accession numbers 5EFF, 5E58, 5EFD, and 5EBA respectively. Abbreviations BSX, xylanase from Bacilllus sp. NG-27; RBSX, recombinant BSX xylanase; TIM, Triosephosphate isomerase; GH10, Glycosyl hydrolase family 10; 3D, three-dimensional; r.m.s.d, root mean square deviation; RSA, relative solvent accessible surface area; Tm, melting temperature; CD, Circular Dichroism; BHX, GH10 xylanase from Bacillus halodurans; BFX, GH10 xylanase from Bacillus firmus; TmxB, GH10 xylanase from Thermotoga maritima