Evolutionary and structural analyses of heterodimeric proteins composed of subunits with same fold

Heterodimeric proteins with homologous subunits of same fold are involved in various biological processes. The objective of this study is to understand the evolution of structural and functional features of such heterodimers. Using a non‐redundant dataset of 70 such heterodimers of known 3D structure and an independent dataset of 173 heterodimers from yeast, we note that the mean sequence identity between interacting homologous subunits is only 23–24% suggesting that, generally, highly diverged paralogues assemble to form such a heterodimer. We also note that the functional roles of interacting subunits/domains are generally quite different. This suggests that, though the interacting subunits/domains are homologous, the high evolutionary divergence characterize their high functional divergence which contributes to a gross function for the heterodimer considered as a whole. The inverse relationship between sequence identity and RMSD of interacting homologues in heterodimers is not followed. We also addressed the question of formation of homodimers of the subunits of heterodimers by generating models of fictitious homodimers on the basis of the 3D structures of the heterodimers. Interaction energies associated with these homodimers suggests that, in overwhelming majority of the cases, such homodimers are unlikely to be stable. Majority of the homologues of heterodimers of known structures form heterodimers (51.8%) and a small proportion (14.6%) form homodimers. Comparison of 3D structures of heterodimers with homologous homodimers suggests that interfacial nature of residues is not well conserved. In over 90% of the cases we note that the interacting subunits of heterodimers are co‐localized in the cell. Proteins 2015; 83:1766–1786. © 2015 Wiley Periodicals, Inc.     

Resolving protein structure‐function‐binding site relationships from a binding site similarity network perspective

Functional annotation is seldom straightforward with complexities arising due to functional divergence in protein families or functional convergence between non‐homologous protein families, leading to mis‐annotations. An enzyme may contain multiple domains and not all domains may be involved in a given function, adding to the complexity in function annotation. To address this, we use binding site information from bound cognate ligands and catalytic residues, since it can help in resolving fold‐function relationships at a finer level and with higher confidence. A comprehensive database of 2,020 fold‐function‐binding site relationships has been systematically generated. A network‐based approach is employed to capture the complexity in these relationships, from which different types of associations are deciphered, that identify versatile protein folds performing diverse functions, same function associated with multiple folds and one‐to‐one relationships. Binding site similarity networks integrated with fold, function, and ligand similarity information are generated to understand the depth of these relationships. Apart from the observed continuity in the functional site space, network properties of these revealed versatile families with topologically different or dissimilar binding sites and structural families that perform very similar functions. As a case study, subtle changes in the active site of a set of evolutionarily related superfamilies are studied using these networks. Tracing of such similarities in evolutionarily related proteins provide clues into the transition and evolution of protein functions. Insights from this study will be helpful in accurate and reliable functional annotations of uncharacterized proteins, poly‐pharmacology, and designing enzymes with new functional capabilities. Proteins 2017; 85:1319–1335. © 2017 Wiley Periodicals, Inc.     

Enhanced functional and structural domain assignments using remote similarity detection procedures for proteins encoded in the genome of<Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> H37Rv

The sequencing of theMycobacterium tuberculosis (MTB) H37Rv genome has facilitated deeper insights into the biology of MTB, yet the functions of many MTB proteins are unknown. We have used sensitive profile-based search procedures to assign functional and structural domains to infer functions of gene products encoded in MTB. These domain assignments have been made using a compendium of sequence and structural domain families. Functions are predicted for 78% of the encoded gene products. For 69% of these, functions can be inferred by domain assignments. The functions for the rest are deduced from their homology to proteins of known function. Superfamily relationships between families of unknown and known structures have increased structural information by ∼ 11%. Remote similarity detection methods have enabled domain assignments for 1325 ‘hypothetical proteins’. The most populated families in MTB are involved in lipid metabolism, entry and survival of the bacillus in host. Interestingly, for 353 proteins, which we refer to as MTB-specific, no homologues have been identified. Numerous, previously unannotated, hypothetical proteins have been assigned domains and some of these could perhaps be the possible chemotherapeutic targets. MTB-specific proteins might include factors responsible for virulence. Importantly, these assignments could be valuable for experimental endeavors. The detailed results are publicly available at http://hodgkin.mbu.iisc.ernet.in/∼dots. An erratum to this article is available at .     

Loss of ABCA8B decreases myelination by reducing oligodendrocyte precursor cells in mice

The myelin sheath, which is wrapped around axons, is a lipid-enriched structure produced by mature oligodendrocytes. Disruption of the myelin sheath is observed in several neurological diseases, such as multiple sclerosis. A crucial component of myelin is sphingomyelin, levels of which can be increased by ABCA8, a member of the ATP-binding cassette transporter family. ABCA8 is highly expressed in the cerebellum, specifically in oligodendroglia. However, whether ABCA8 plays a role in myelination and mechanisms that would underlie this role remain unknown. Here, we found that the absence of Abca8b, a mouse ortholog of ABCA8, led to decreased numbers of cerebellar oligodendrocyte precursor cells (OPCs) and mature oligodendrocytes in mice. We show that in oligodendrocytes, ABCA8 interacts with chondroitin sulfate proteoglycan 4 (CSPG4), a molecule essential for OPC proliferation, migration, and myelination. In the absence of Abca8b, localization of CSPG4 to the plasma membrane was decreased, contributing to reduced cerebellar CSPG4 expression. Cerebellar CSPG4+ OPCs were also diminished, leading to decreased mature myelinating oligodendrocyte numbers and cerebellar myelination levels in Abca8b^?/? mice. In addition, electron microscopy analyses showed that the number of nonmyelinated cerebellar axons was increased, whereas cerebellar myelin thickness (g-ratio), myelin sheath periodicity, and axonal diameter were all decreased, indicative of disordered myelin ultrastructure. In line with disrupted cerebellar myelination, Abca8b^?/? mice showed lower cerebellar conduction velocity and disturbed locomotion. In summary, ABCA8 modulates cerebellar myelination, in part through functional regulation of the ABCA8-interacting protein CSPG4. Our findings suggest that ABCA8 disruption may contribute to the pathophysiology of myelin disorders.     

Synthesis of 2-deoxy-D-arabino/lyxo-hexopyranosyl disaccharides

Synthesis of 2-deoxy-D-arabino/lyxo-hexopyranosyl disaccharides is reported. In these, the disaccharides contain 2-deoxy-arabino-hexopyranosyl and 2-deoxy-lyxo-hexopyranosyl sugars as either the reducing or the non-reducing or both the sugar units of the disaccharides. The activated 2-deoxy-1-thioglycosides served as the common precursors to prepare the 2-deoxy disaccharides with the above configurations.     

DNA recognition by Zn [Cysteinyl-His-OMe]2: A minimal zinc finger docking unit

Zinc fingers, 30-residue peptides anchored on Zn(II) coordinated to pairs of cysteines and histidines, recognize DNA triplets and, as tandem modules, effect sequence read out. The focus of zinc finger-DNA interaction studies thus far has been to probe the nature of the binding of the 12-residue recognition element of the finger with DNA code bases. To understand the possible role of the Zn(II) ligand and to assess its own DNA interaction profile, [(CH)₂Zn] (C: cysteine; H: histidine; Figure 1) was constructed from bis-^t Boc-cystinyl-di-His-OMe via thiol-disulfide exchange, Zn(II) complexation, and deprotection. [(CH)₂Zn] binds with polyd- (G·C)·polyd(G·C) with association constants—1.8 × 10⁷ M⁻¹ (specific DNA-phosphate) and 3.3 × 10³ M⁻¹ (nonspecific DNA-phosphate); perturbs its B-DNA profile; and enhances the T_m from 62.5 to 70.15°C in a concentration-independent manner, with an ideal reversal profile on cooling, not observed in the DNA alone; releases polyd(G·C)·polyd(G·C)-bound ethidium bromide; enhances the fluorescence of polyd(G·C)·polyd(G·C)-bound ethidium bromide at low concentrations; and quenches it at higher ranges. [(CH)₂Zn] also binds to d(ACGCTGGGCGT), the sequence associated with Zif-268, 3-finger binding site. Such interactions were not seen in parallel studies with (a) polyd(A·T)·polyd(A·T) and [(CH)₂Zn] and (b) {[C′H₂] (C′: cystine; H: histidine; the direct metal-free precursor of [(CH)₂Zn]}, ionic zinc nitrate, and covalent zinc acetyl acetonate Zn(AcAc)₂, with poly[d(G·C)·polyd(G·C)]. The results are rationalized on the basis of two types of association between [(CH)₂Zn] and polyd(G·C)·polyd(G·C), a nonspecific recognition of the sugar phosphate backbone, by an imidazole of [(CH)₂Zn] and a specific one involving the amino group of [(CH)₂Zn] and the guanine base of DNA. Control experiments show that the latter greatly promotes DNA recognition. The possibility for such specific interactions with relatively small structures of the type [(CH)₂Zn] would be of use in the design of DNA recognition elements and also provide an explanation for the experimentally found variation in the placement of the zinc finger docking unit around the major groove of DNA. © 1997 John Wiley & Sons, Inc.     

WASP-interacting protein (WIP): working in polymerisation and much more

The migration of cells and the movement of some intracellular pathogens, such as Shigella and Vaccinia, are dependent on the actin-based cytoskeleton. Many proteins are involved in regulating the dynamics of the actin-based microfilaments within cells and, among them, WASP and N-WASP have a significant role in the regulation of actin polymerisation. The activity and stability of WASP is regulated by its cellular partner WASP-interacting protein (WIP) during the formation of actin-rich structures, including the immune synapse, filopodia, lamellipodia, stress fibres and podosomes. Here, we review the role of WIP in regulating WASP function by stabilising WASP and shuttling WASP to areas of actin assembly in addition to reviewing the WASP-independent functions of WIP.     

Total synthesis of a depsidomycin analogue by convergent solid‐phase peptide synthesis and macrolactonization strategy for antitubercular activity

Depsidomycin is a cyclic heptadepsi‐peptide isolated from the cultured broth of Streptomyces lavendofoliae MI951‐62F2. It exhibits significant antimicrobial and immunosuppressive activity. The total synthesis of a depsidomycin analogue in which 1,2‐piperazine‐3‐carboxylic acid was substituted with proline is described. After several trials using different strategies, the desired depsidomycin analogue was obtained via stepwise synthesis starting by the amino acid ‘head’ and macrolactonization under Yamaguchi conditions. The cyclic depsipeptide was evaluated to have an minimum inhibitory concentration (MIC) of 4 µg/ml against H37RV and 16 µg/ml against MDR clinical strains of MTB (MDR‐MTB), while the linear precursor 8 also had MICs of 4 and 16 µg/ml for the susceptible and resistant strains, respectively. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Conservation of structural fluctuations in homologous protein kinases and its implications on functional sites

Our aim is to explore the similarities in structural fluctuations of homologous kinases. Gaussian Network Model based Normal Mode Analysis was performed on 73 active conformation structures in Ser/Thr/Tyr kinase superfamily. Categories of kinases with progressive evolutionary divergence, viz. (i) Same kinase with many crystal structures, (ii) Within‐Subfamily, (iii) Within‐Family, (iv) Within‐Group, and (v) Across‐Group, were analyzed. We identified a flexibility signature conserved in all kinases involving residues in and around the catalytic loop with consistent low‐magnitude fluctuations. However, the overall structural fluctuation profiles are conserved better in closely related kinases (Within‐Subfamily and Within‐family) than in distant ones (Within‐Group and Across‐Group). A substantial 65.4% of variation in flexibility was not accounted by variation in sequences or structures. Interestingly, we identified substructural residue‐wise fluctuation patterns characteristic of kinases of different categories. Specifically, we recognized statistically significant fluctuations unique to families of protein kinase A, cyclin‐dependent kinases, and nonreceptor tyrosine kinases. These fluctuation signatures localized to sites known to participate in protein‐protein interactions typical of these kinase families. We report for the first time that residues characterized by fluctuations unique to the group/family are involved in interactions specific to the group/family. As highlighted for Src family, local regions with differential fluctuations are proposed as attractive targets for drug design. Overall, our study underscores the importance of consideration of fluctuations, over and above sequence and structural features, in understanding the roles of sites characteristic of kinases. Proteins 2016; 84:957–978. © 2016 Wiley Periodicals, Inc.     

Microbial Response to Organic Matter Enrichment in the Oligotrophic Levantine Basin (Eastern Mediterranean)

The response of a deep-water benthic microbial community to organic matter (OM) enrichment was studied in the unexplored region of the SW Cretan margin (E. Mediterranean). A food pulse of 0.5 g C m^?2 was simulated by adding ¹³C-labelled diatoms to sediment cores retrieved from 1079 m depth. The diatom addition resulted in a significant increase in the sediment community oxygen consumption (SCOC). After 6 days, ～50 mg C m^?2 of the added material was processed by the microbial community. The major carbon sink was respiration, which accounted for ～96% of the total processed material. The carbon uptake rate (12 mg C m^?2 d^?1) was considerably lower than previously published values in the E. Mediterranean at similar depths. The microbial community in our study site is distinct, as revealed by the unusually high presence of branched phospholipid fatty acids (PLFA). Previous studies have revealed that the slope under investigation may act as a conduit of OM from the shallow shelf to the deep basins, resulting in the prevalence of relatively refractory OM at mid-slope depths. We postulate that sedimentary processes affect the amount of bioavailable sedimentary OM and consequently the structure and physiological state of bacterial community in our study site. The distinct microbial community composition at our station compared to more stable adjacent slopes could explain the limited response of the microbial community to the addition of labile OM. Supplemental materials are available for this article. Go to the publisher's online edition of Geomicrobiology Journal to view the free supplemental file.