The Mycobacterium tuberculosis methyltransferase Rv2067c manipulates host epigenetic programming to promote its own survival.

Mycobacterium tuberculosis has evolved several mechanisms to counter host defense arsenals for its proliferation. Here we report that M. tuberculosis employs a multi-pronged approach to modify host epigenetic machinery for its survival. It secretes methyltransferase (MTase) Rv2067c into macrophages, trimethylating histone H3K79 in a non-nucleosomal context. Rv2067c downregulates host MTase DOT1L, decreasing DOT1L-mediated nucleosomally added H3K79me3 mark on pro-inflammatory response genes. Consequent inhibition of caspase-8-dependent apoptosis and enhancement of RIPK3-mediated necrosis results in increased pathogenesis. In parallel, Rv2067c enhances the expression of SESTRIN3, NLRC3, and TMTC1, enabling the pathogen to overcome host inflammatory and oxidative responses. We provide the structural basis for differential methylation of H3K79 by Rv2067c and DOT1L. The structures of Rv2067c and DOT1L explain how their action on H3K79 is spatially and temporally separated, enabling Rv2067c to effectively intercept the host epigenetic circuit and downstream signaling.

Homology Modeling of Antibody Variable Regions: Methods and Applications.

Adaptive immunity specifically protects us from antigenic challenges. Antibodies are key effector proteins of adaptive immunity, and they are remarkable in their ability to recognize a virtually limitless number of antigens. Fragment variable (FV), the antigen-binding region of antibodies, can be split into two main components, namely, framework and complementarity determining regions. The framework (FR) consists of light-chain framework (FRL) and heavy-chain framework (FRH). Similarly, the complementarity determining regions (CDRs) comprises of light-chain CDRs 1-3 (CDRs L1-3) and heavy-chain CDRs 1-3 (CDRs H1-3). While FRs are relatively constant in sequence and structure across diverse antibodies, sequence variation in CDRs leading to differential conformations of CDR loops accounts for the distinct antigenic specificities of diverse antibodies. The conserved structural features in FRs and conformity of CDRs to a limited set of standard conformations allow for the accurate prediction of FV models using homology modeling techniques. Antibody structure prediction from its amino acid sequence has numerous important applications including prediction of antibody-antigen interaction interfaces and redesign of therapeutically and biotechnologically useful antibodies with improved affinity. This chapter summarizes the current practices employed in the successful homology modeling of antibody variable regions and the potential applications of the generated homology models.

Targeted Redesign of Suramin Analogs for Novel Antimicrobial Lead Development.

The emergence of new viral infections and drug-resistant bacteria urgently necessitates expedient therapeutic development. Repurposing and redesign of existing drugs against different targets are one potential way in which to accelerate this process. Suramin was initially developed as a successful antiparasitic drug but has also shown promising antiviral and antibacterial activities. However, due to its high conformational flexibility and negative charge, suramin is considered quite promiscuous toward positively charged sites within nucleic acid binding proteins. Although some suramin analogs have been developed against specific targets, only limited structure-activity relationship studies were performed, and virtual screening has yet to be used to identify more specific inhibitor(s) based on its scaffold. Using available structures, we investigated suramin's target diversity, confirming that suramin preferentially binds to protein pockets that are both positively charged and enriched in aromatic or leucine residues. Further, suramin's high conformational flexibility allows adaptation to structurally diverse binding surfaces. From this platform, we developed a framework for structure- and docking-guided elaboration of suramin analog scaffolds using virtual screening of suramin and heparin analogs against a panel of diverse therapeutically relevant viral and bacterial protein targets. Use of this new framework to design potentially specific suramin analogs is exemplified using the SARS-CoV-2 RNA-dependent RNA polymerase and nucleocapsid protein, identifying leads that might inhibit a wide range of coronaviruses. The approach presented here establishes a computational framework for designing suramin analogs against different bacterial and viral targets and repurposing existing drugs for more specific inhibitory activity.

Small Glycols Discover Cryptic Pockets on Proteins for Fragment-Based Approaches.

Cryptic pockets are visible in ligand-bound protein structures but are occluded in unbound structures. Utilizing these pockets in fragment-based drug-design provides an attractive option for proteins not tractable by classical binding sites. However, owing to their hidden nature, they are difficult to identify. Here, we show that small glycols find cryptic pockets on a diverse set of proteins. Initial crystallography experiments serendipitously revealed the ability of ethylene glycol, a small glycol, to identify a cryptic pocket on the W6A mutant of the RBSX protein (RBSX-W6A). Explicit-solvent molecular dynamics (MD) simulations of RBSX-W6A with the exposed state of the cryptic pocket (ethylene glycol removed) revealed closure of the pocket reiterating that the exposed state of cryptic pockets in general are unstable in the absence of ligands. Also, no change in the pocket was observed for simulations of RBSX-W6A with the occluded state of the cryptic pocket, suggesting that water molecules are not able to open the cryptic pocket. "Cryptic-pocket finding" potential of small glycols was then supported and generalized through additional crystallography experiments, explicit-cosolvent MD simulations, and protein data set construction and analysis. The cryptic pocket on RBSX-W6A was found again upon repeating the crystallography experiments with another small glycol, propylene glycol. Use of ethylene glycol as a probe molecule in cosolvent MD simulations led to the enhanced sampling of the exposed state of experimentally observed cryptic sites on a test set of two proteins (Niemann-Pick C2, Interleukin-2). Further, analyses of protein structures with validated cryptic sites showed that ethylene glycol molecules bind to sites on proteins (Bcl-xL, G-actin, myosin II, and glutamate receptor 2), which become apparent upon binding of biologically relevant ligands. Our study thus suggests potential application of the small glycols in experimental and computational fragment-based approaches to identify cryptic pockets in apparently undruggable and/or difficult targets, making these proteins amenable to drug-design strategies.

Interrogation of 3D-swapped structure and functional attributes of quintessential Sortase A from Streptococcus pneumoniae.

The anchoring of the surface proteins to the cell wall in gram-positive bacteria involves a peptide ligation reaction catalyzed by transpeptidase sortase. Most bacterial genomes encode multiple sortases with dedicated functions. Streptococcus pneumoniae (Sp) carries four sortases; a housekeeping sortase (SrtA), and three pilin specific sortases (SrtC1, C2, C3) dedicated to the biosynthesis of covalent pilus. Interestingly, SrtA, meant for performing housekeeping roles, is also implicated in pilus assembly of Sp. The allegiance of SpSrtA to the pathogenic pilus assembly makes it an ideal target for clinical inhibitor development. In this paper, we describe biochemical characterization, crystal structure and peptide substrate preference of SpSrtA. Transpeptidation reaction with a variety of substrates revealed that the enzyme preferred elongated LPXTG sequences and transferred them equally well to both Ala- and Gly-terminated peptides. Curiously, crystal structure of both wild type and an active site (Cys to Ala) mutant of SpSrtA displayed inter-twined 3D-swapped dimers in which each protomer generated a classic eight stranded beta-barrel "sortase fold". Size-exclusion chromatography and sedimentation equilibrium measurements revealed predominant presence of a dimer in equilibrium with its monomer. The crystal structure-based Cys-Cys distance mapping with defined chemical cross-linkers established the existence of 3D-swapped structure in solution. The swapping in SpSrtA, unprecedented for sortase family, may be physiologically relevant and meant to perform regulatory functions.

Structural basis for neutralization of cytotoxic abrin by monoclonal antibody D6F10.

Abrin, an extremely cytotoxic Type II ribosome-inactivating protein (RIP), is a potential bio-warfare agent. Abrin A-chain (ABA) depurinates an adenosine of sarcin-ricin loop (SRL) from eukaryotic 28S rRNA, thereby arresting protein synthesis and leading to cell death. Monoclonal antibody (mAb) D6F10 is the only known antibody that neutralizes ABA's activity in cell-free systems as well as abrin's toxicity in vitro and in vivo. However, how binding of mAb D6F10 to abrin interferes with abrin's catalytic activity at ribosome is still poorly understood. To provide structural basis for mAb D6F10-mediated rescue of ribosome inactivation by abrin, we determined crystal structures of ABA with and without substrate analogs. The structures of ABA-substrate analogs and ribosome were used in an experiment-guided computational protocol, to construct the ABA-Ribosome complex. A homology model of the variable region (Fv ) of mAb D6F10 was generated and docked with the apo-ABA structure to construct the ABA-D6F10 Fv complex. Structural superposition of ABA common to ABA-D6F10 Fv and ABA-Ribosome complexes reveals steric hindrance as the primary mechanism by which mAb D6F10 neutralizes abrin. In contrast to ABA alone, ABA bound to mAb D6F10 is unable to access the SRL on the ribosome owing to steric clashes of mAb D6F10 with the ribosome. Crystal structures of ABA also reveal a catalytic water molecule implicated in hydrolyzing N-glycosidic bond of the susceptible adenosine by RIPs. Furthermore, our strategy provides structural details of steric hindrance important for neutralization of ricin, another RIP, by mAb 6C2 and hence is of wide applicability. ENZYME: EC3.2.2.22. DATABASE: Structural data have been deposited in the Protein Data Bank (PDB) under the accession numbers 5Z37, 5Z3I, and 5Z3J.

l-Dopa and dopamine conjugated naphthalenediimides modulate amyloid ß toxicity.

The process of protein misfolding and aggregation to form neurotoxic species is strongly implicated in most of the neurodegenerative disorders. In particular, amyloid beta (Aß) misfolding and aggregation is central to pathophysiological processes of Alzheimer's disease. The development of aggregation modulators has enormous implications in the discovery of effective therapeutic agents for Alzheimer's disease. Herein, we report the design and synthesis of a series of natural amino acid, l-dopa and dopamine appended derivatives of naphthalenediimide (NDI) to identify efficient aggregation modulators. Furthermore, the molecular docking studies revealed the possible binding sites and binding mode of NDI-conjugates to Aß aggregates. Among the designed NDI-conjugates, l-dopa and dopamine derivatives (NLD and NDP, respectively) showed excellent aggregation modulation efficiency (inhibition and dissolution), as shown by the thioflavin T (ThT) binding assays, dot blot analysis and in cellulo studies. The docking results from in silico studies are in good agreement with the experimental data. In addition to their significant modulation efficiency towards Aß aggregation, NLD and NDP possess antioxidant activity conducive to the development of disease-modifying therapeutic agents for the treatment of Alzheimer's disease.

Conformational heterogeneity in tails of DNA-binding proteins is augmented by proline containing repeats.

A cationic terminal extension or tail is a common feature of many DNA-binding proteins. We show that a particular type of tail rich in proline, alanine and lysine belongs to the class of 'flexible disorder' and consists of characteristic pentapeptide repeats. Our designed peptides, (AAKKA)1-4 and (PAKKA)1-4, represent the tails of several bacterial DNA-binding proteins. Enhanced conformational sampling of these representative peptides using accelerated molecular dynamic simulations supported by circular dichroism spectroscopy and nuclear magnetic resonance studies demonstrates the role of frequent and interspersed prolines in augmenting conformational heterogeneity of the peptide backbone. Analysis of circular variance of backbone dihedral angles indicates alternating regions of relative rigidity and flexibility along the peptide sequence due to prolines. Preferred placement of lysines in the regions of higher backbone flexibility might improve DNA-binding by conformational selection. Our results could be relevant for rational de novo design of disordered peptides.

Molecular Dynamics Information Improves cis-Peptide-Based Function Annotation of Proteins.

cis-Peptide bonds, whose occurrence in proteins is rare but evolutionarily conserved, are implicated to play an important role in protein function. This has led to their previous use in a homology-independent, fragment-match-based protein function annotation method. However, proteins are not static molecules; dynamics is integral to their activity. This is nicely epitomized by the geometric isomerization of cis-peptide to trans form for molecular activity. Hence we have incorporated both static (cis-peptide) and dynamics information to improve the prediction of protein molecular function. Our results show that cis-peptide information alone cannot detect functional matches in cases where cis-trans isomerization exists but 3D coordinates have been obtained for only the trans isomer or when the cis-peptide bond is incorrectly assigned as trans. On the contrary, use of dynamics information alone includes false-positive matches for cases where fragments with similar secondary structure show similar dynamics, but the proteins do not share a common function. Combining the two methods reduces errors while detecting the true matches, thereby enhancing the utility of our method in function annotation. A combined approach, therefore, opens up new avenues of improving existing automated function annotation methodologies.

Structure and specificity of a new class of Ca2+-independent housekeeping sortase from Streptomyces avermitilis provide insights into its non-canonical substrate preference.

Surface proteins in Gram-positive bacteria are incorporated into the cell wall through a peptide ligation reaction catalyzed by transpeptidase sortase. Six main classes (A-F) of sortase have been identified of which class A sortase is meant for housekeeping functions. The prototypic housekeeping sortase A (SaSrtA) from Staphylococcus aureus cleaves LPXTG-containing proteins at the scissile T-G peptide bond and ligates protein-LPXT to the terminal Gly residue of the nascent cross-bridge of peptidoglycan lipid II precursor. Sortase-mediated ligation ("sortagging") of LPXTG-containing substrates and Gly-terminated nucleophiles occurs in vitro as well as in cellulo in the presence of Ca2+ and has been applied extensively for protein conjugations. Although the majority of applications emanate from SaSrtA, low catalytic efficiency, LPXTG specificity restriction, and Ca2+ requirement (particularly for in cellulo applications) remain a drawback. Given that Gram-positive bacteria genomes encode a variety of sortases, natural sortase mining can be a viable complementary approach akin to engineering of wild-type SaSrtA. Here, we describe the structure and specificity of a new class E sortase (SavSrtE) annotated to perform housekeeping roles in Streptomyces avermitilis Biochemical experiments define the attributes of an optimum peptide substrate, demonstrate Ca2+-independent activity, and provide insights about contrasting functional characteristics of SavSrtE and SaSrtA. Crystal structure, substrate docking, and mutagenesis experiments have identified a critical residue that dictates the preference for a non-canonical LAXTG recognition motif over LPXTG. These results have implications for rational tailoring of substrate tolerance in sortases. Besides, Ca2+-independent orthogonal specificity of SavSrtE is likely to expand the sortagging toolkit.

Structural and evolutionary analyses reveal determinants of DNA binding specificities of nucleoid-associated proteins HU and IHF.

Nucleoid-associated proteins (NAPs) are chromosome-organizing factors, which affect the transcriptional landscape of a bacterial cell. HU is an NAP, which binds to DNA with a broad specificity while homologous IHF (Integration Host Factor), binds DNA with moderately higher specificity. Specificity and differential binding affinity of HU/IHF proteins towards their target binding sites play a crucial role in their regulatory dynamics. Decades of biochemical and genomic studies have been carried out for HU and IHF like proteins. Yet, questions related to their DNA binding specificity, and differential ability to bend DNA thus affecting the binding site length remained unanswered. In addition, the problem has not been investigated from an evolutionary perspective. Our phylogenetic analysis revealed three major clades belonging to HU, IHFα and IHFß like proteins with reference to E. coli. We carried out a comparative analysis of three-dimensional structures of HU/IHF proteins to gain insight into the structural basis of clade division. The present study revealed three major features which contribute to differential DNA binding specificity of HU/IHF proteins, (I) conformational restriction of DNA binding residues due to salt-bridge formation, (II) the enrichment of alanine in the DNA binding site increasing conformational space of flexible side chains in its vicinity and (III) nature of DNA binding residue (Arg to Lys bias in different clades) which interacts differentially to DNA bases. We observed an extended electropositive surface at the DNA draping site for IHF clade proteins compared to HU, which stabilizes the DNA bend. Differences in the dimer stabilization strategies between HU and IHF were also observed. Our analysis reveals a comprehensive evolutionary picture, which rationalizes the origin of multi-specificity of HU/IHF proteins using sequence and structure-based determinants, which could also be applied to understand differences in binding specificities of other nucleic acid binding proteins.

Vesicular Nanostructure Formation by Self-Assembly of Anisotropic Penta-phenol-Substituted Fullerene in Water.

A study on self-assembly of anisotropically substituted penta-aryl fullerenes in water has been reported. The penta-phenol-substituted amphiphilic fullerene derivative [C60Ph5(OH)5] exhibited self-assembled vesicular nanostructures in water under the experimental conditions. The size of the vesicles was observed to depend upon the kinetics of self-assembly and could be varied from ∼300 to ∼70 nm. Our mechanistic study indicated that the self-assembly of C60Ph5(OH)5 is driven by extensive intermolecular as well as water-mediated hydrogen bonding along with fullerene-fullerene hydrophobic interaction in water. The cumulative effect of these interactions is responsible for the stability of vesicular structures even on the removal of solvent. The substitution of phenol with anisole resulted in different packing and interaction of the fullerene derivative, as indicated in the molecular dynamics studies, thus resulting in different self-assembled nanostructures. The hollow vesicles were further encapsulated with a hydrophobic conjugated polymer and water-soluble dye as guest molecules. Such confinement of π-conjugated polymers in fullerene has significance in bulk heterojunction devices for efficient exciton diffusion.

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

UNLABELLED: 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 (ß/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. DATABASE: The coordinates of RBSX, V1A and V1L have been deposited in the PDB database under the accession numbers 4QCE, 4QCF, and 4QDM, respectively.

Crystallographic insight-guided nanoarchitectonics and conductivity modulation of an n-type organic semiconductor through peptide conjugation.

Crystallographic insight-guided nanoarchitectonics of peptide-conjugated naphthalene diimide (NDI) is described. In a bio-inspired approach, non-proteinogenic α-amino isobutyric acid (Aib)- and alanine (Ala)-derived peptides orchestrated the 1D achiral and 2D chiral molecular ordering of NDI, respectively, which resulted in modulation of nanoscale morphology, chiroptical and conductivity properties.

Mycobacterium tuberculosis cell division protein, FtsE, is an ATPase in dimeric form.

FtsE is one of the earliest cell division proteins that assembles along with FtsX at the mid-cell site during cell division in Escherichia coli. Both these proteins are highly conserved across diverse bacterial genera and are predicted to constitute an ABC transporter type complex, in which FtsE is predicted to bind ATP and hydrolyse it, and FtsX is predicted to be an integral membrane protein. We had earlier reported that the MtFtsE of the human pathogen, Mycobacterium tuberculosis, binds ATP and interacts with MtFtsX on the cell membrane of M. tuberculosis and E. coli. In this study, we demonstrate that MtFtsE is an ATPase, the active form of which is a dimer, wherein the participating monomers are held together by non-covalent interactions, with the Cys84 of each monomer present at the dimer interface. Under oxidising environment, the dimer gets stabilised by the formation of Cys84-Cys84 disulphide bond. While the recombinant MtFtsE forms a dimer on the membrane of E. coli, the native MtFtsE seems to be in a different conformation in the M. tuberculosis membrane. Although disulphide bridges were not observed on the cytoplasmic side (reducing environment) of the membrane, the two participating monomers could be isolated as dimers held together by non-covalent interactions. Taken together, these findings show that MtFtsE is an ATPase in the non-covalent dimer form, with the Cys84 of each monomer present in the reduced form at the dimer interface, without participating in the dimerisation or the catalytic activity of the protein.

Identifying functionally important cis-peptide containing segments in proteins and their utility in molecular function annotation.

Cis-peptide embedded segments are rare in proteins but often highlight their important role in molecular function when they do occur. The high evolutionary conservation of these segments illustrates this observation almost universally, although no attempt has been made to systematically use this information for the purpose of function annotation. In the present study, we demonstrate how geometric clustering and level-specific Gene Ontology molecular-function terms (also known as annotations) can be used in a statistically significant manner to identify cis-embedded segments in a protein linked to its molecular function. The present study identifies novel cis-peptide fragments, which are subsequently used for fragment-based function annotation. Annotation recall benchmarks interpreted using the receiver-operator characteristic plot returned an area-under-curve > 0.9, corroborating the utility of the annotation method. In addition, we identified cis-peptide fragments occurring in conjunction with functionally important trans-peptide fragments, providing additional insights into molecular function. We further illustrate the applicability of our method in function annotation where homology-based annotation transfer is not possible. The findings of the present study add to the repertoire of function annotation approaches and also facilitate engineering, design and allied studies around the cis-peptide neighborhood of proteins.

Targeting Mycobacterium tuberculosis nucleoid-associated protein HU with structure-based inhibitors.

The nucleoid-associated protein HU plays an important role in maintenance of chromosomal architecture and in global regulation of DNA transactions in bacteria. Although HU is essential for growth in Mycobacterium tuberculosis (Mtb), there have been no reported attempts to perturb HU function with small molecules. Here we report the crystal structure of the N-terminal domain of HU from Mtb. We identify a core region within the HU-DNA interface that can be targeted using stilbene derivatives. These small molecules specifically inhibit HU-DNA binding, disrupt nucleoid architecture and reduce Mtb growth. The stilbene inhibitors induce gene expression changes in Mtb that resemble those induced by HU deficiency. Our results indicate that HU is a potential target for the development of therapies against tuberculosis.