ARL15, a GTPase implicated in rheumatoid arthritis, potentially repositions its truncated N-terminus as a function of guanine nucleotide binding.

The ADP ribosylation factor like protein 15 (ARL15) gene encodes for an uncharacterized GTPase associated with rheumatoid arthritis (RA) and other metabolic disorders. Investigation of the structural and functional attributes of ARL15 is important to position the protein as a potential drug target. Using spectroscopy, we demonstrated that ARL15 exhibits properties inherent of GTPases. The Km and Vmax of the enzyme were calculated to be 100 µM and 1.47 µmole/min/µL, respectively. The equilibrium dissociation constant (Kd) of GTP binding with ARL15 was estimated to be about eight-fold higher than that of GDP. Small Angle X-ray Scattering (SAXS) data indicated that in solution, the apo state of monomeric ARL15 adopts a shape characterized by a globe of maximum linear dimension (Dmax) of 6.1 nm, and upon binding to GTP or GDP, the vector distribution profile changes to peak-n-tail shoulder with Dmax extended to 7.6 and 7.7 nm, respectively. Structure restoration using a sequence-based template and experimental SAXS data provided the first visual insight revealing that the folded N-terminal in the unbound state of the protein may toggle open upon binding to guanine nucleotides. The conformational dynamics observed in the N-terminal region offer a scope to develop drugs that target this unique GTPase, potentially providing treatments for a range of metabolic disorders.

Insights into dynamic changes in ADC-7 and P99 cephalosporinases using small angle x-ray scattering (SAXS).

To counter the emergence of ß-lactamase (BL) mediated resistance, design of new ß-lactamase inhibitors (BLIs) is critical. Many high-resolution crystallographic structures of BL complexed with BLIs are available. However, their impact on BLI design is struggling to keep pace with novel and emerging variants. Small angle x-ray scattering (SAXS) in combination with molecular modeling is a useful tool to determine dynamic structures of macromolecules in solution. An important application of SAXS is to determine the conformational changes that occur when BLI bind to BL. To probe if conformational dynamics occur in class C cephalosporinases, we studied SAXS profiles of two clinically relevant class C ß-lactamases, Acinetobacter baumannii ADC-7 and Enterobacter cloacae P99 in apo format complexed with BLIs. Importantly, SAXS data analysis demonstrated that in solution, these representative class C enzymes remain monomeric and did not show the associated assemblies that were seen in various crystal structures. SAXS data acquired for ADC-7 and P99, in apo and inhibitor bound states, clearly showed that these enzymes undergo detectable conformational changes, and these class C ß-lactamases also close upon binding inhibitors as does BlaC. Further analysis revealed that addition of inhibitor led to the compacting of a range of residues around the active site, indicating that the conformational changes that both P99 and ADC-7 undergo are central to inhibitor recognition and efficacy. Our findings support the importance of exploring conformational changes using SAXS analysis in the design of future BLIs.Communicated by Ramaswamy H. Sarma.

Structural and Functional Characterization of Rv0792c from Mycobacterium tuberculosis: Identifying Small Molecule Inhibitor against HutC Protein.

In order to adapt in host tissues, microbial pathogens regulate their gene expression through a variety of transcription factors. Here, we have functionally characterized Rv0792c, a HutC homolog from Mycobacterium tuberculosis. In comparison to the parental strain, a strain of M. tuberculosis with a Rv0792c mutant was compromised for survival upon exposure to oxidative stress and infection in guinea pigs. RNA sequencing analysis revealed that Rv0792c regulates the expression of genes involved in stress adaptation and virulence of M. tuberculosis. Solution small-angle X-ray scattering (SAXS) data-steered model building confirmed that the C-terminal region plays a pivotal role in dimer formation. Systematic evolution of ligands by exponential enrichment (SELEX) resulted in the identification of single-strand DNA (ssDNA) aptamers that can be used as a tool to identify small-molecule inhibitors targeting Rv0792c. Using SELEX and SAXS data-based modeling, we identified residues essential for Rv0792c's aptamer binding activity. In this study, we also identified I-OMe-Tyrphostin as an inhibitor of Rv0792c's aptamer and DNA binding activity. The identified small molecule reduced the growth of intracellular M. tuberculosis in macrophages. The present study thus provides a detailed shape-function characterization of a HutC family of transcription factor from M. tuberculosis. IMPORTANCE Prokaryotes encode a large number of GntR family transcription factors that are involved in various fundamental biological processes, including stress adaptation and pathogenesis. Here, we investigated the structural and functional role of Rv0792c, a HutC homolog from M. tuberculosis. We demonstrated that Rv0792c is essential for M. tuberculosis to adapt to oxidative stress and establish disease in guinea pigs. Using a systematic evolution of ligands by exponential enrichment (SELEX) approach, we identified ssDNA aptamers from a random ssDNA library that bound to Rv0792c protein. These aptamers were thoroughly characterized using biochemical and biophysical assays. Using SAXS, we determined the structural model of Rv0792c in both the presence and absence of the aptamers. Further, using a combination of SELEX and SAXS methodologies, we identified I-OMe-Tyrphostin as a potential inhibitor of Rv0792c. Here we provide a detailed functional characterization of a transcription factor belonging to the HutC family from M. tuberculosis.

Plasmodium falciparum Kelch13 and its artemisinin-resistant mutants assemble as hexamers in solution: a SAXS data-driven modelling study.

The artemisinin-resistant mutations in Plasmodium falciparum (PfKelch13) identified worldwide are mostly confined to the Broad-complex, tramtrack and bric-à-brac/poxvirus and zinc-finger (BTB/POZ) and Kelch-repeat propeller (KRP) domains. To date, only two crystal structures of the BTB/POZ-KRP domains as tight dimers are available, which limits structure-based predictions and interpretation of its role(s) in inducing clinical artemisinin resistance. Our solution Small-Angle X-ray Scattering (SAXS) data analysis and shape restoration brought forth that: (a) PfKelch13 forms a stable hexamer in P6 symmetry, (b) interactions of the N-termini drive the hexameric assembly, and (c) the six KRP domains project independently in space, forming a cauldron-like architecture. We further deduce that the artemisinin-sensitive mutant A578S is packed like the wild-type protein, however, hexameric assemblies of the predominant artemisinin-resistant mutants R539T and C580Y displayed detectable differences in the spatial positioning of their BTB/POZ-KRP domains. Lastly, mapping of mutations known to enable artemisinin resistance suggested evolutionary pressure in the selection for mutations in the BTB/POZ-KRP domains. These mutations appear non-detrimental to the hexameric assembly of proteins, and yet somehow alter the flux of downstream events essential for the susceptibility to artemisinin.

Elicitation of potent serum neutralizing antibody responses in rabbits by immunization with an HIV-1 clade C trimeric Env derived from an Indian elite neutralizer.

Evaluating the structure-function relationship of viral envelope (Env) evolution and the development of broadly cross-neutralizing antibodies (bnAbs) in natural infection can inform rational immunogen design. In the present study, we examined the magnitude and specificity of autologous neutralizing antibodies induced in rabbits by a novel HIV-1 clade C Env protein (1PGE-THIVC) vis-à-vis those developed in an elite neutralizer from whom the env sequence was obtained that was used to prepare the soluble Env protein. The novel 1PGE-THIVC Env trimer displayed a native like pre-fusion closed conformation in solution as determined by small angle X-ray scattering (SAXS) and negative stain electron microscopy (EM). This closed spike conformation of 1PGE-THIVC Env trimers was correlated with weak or undetectable binding of non-neutralizing monoclonal antibodies (mAbs) compared to neutralizing mAbs. Furthermore, 1PGE-THIVC SOSIP induced potent neutralizing antibodies in rabbits to autologous virus variants. The autologous neutralizing antibody specificity induced in rabbits by 1PGE-THIVC was mapped to the C3/V4 region (T362/P401) of viral Env. This observation agreed with electron microscopy polyclonal epitope mapping (EMPEM) of the Env trimer complexed with IgG Fab prepared from the immunized rabbit sera. Our study demonstrated neutralization of sequence matched and unmatched autologous viruses by serum antibodies induced in rabbits by 1PGE-THIVC and also highlighted a comparable specificity for the 1PGE-THIVC SOSIP trimer with that seen with polyclonal antibodies elicited in the elite neutralizer by negative-stain electron microscopy polyclonal epitope (ns-EMPEM) mapping.

Monomeric human soluble CD4 dimerizes at physiological temperature: VTSAXS data based modeling and screening of retardant molecules.

Earlier, solution small angle X-ray scattering (SAXS) data at 10 °C showed that soluble CD4 (sCD4; 1 mg/ml) is monomer with shape similar to single chain in crystal structures of its dimer. Query remained whether the dimeric state of CD4 can form independent of packing effects of crystal? Taking cue from other systems, we explored heat induced possible association of native shapes of sCD4 by variable temperature SAXS (VTSAXS) experiments. The predominant particle size increased consistently with temperature and around 35-40 °C, the estimated mass indicated dimeric state in solution. Furthermore, the observed association was found to be reversible to certain extent. Using SAXS profile representing dimer and crystal structure of monomer, we solved models of CD4 dimers which were dominated by D4-D4 interactions and appeared "wobbling" about the crystal structure of dimeric CD4, convincing pre-existence of crystal-like association in solution. To break the dimerization, we theoretically screened for small molecules binding to dimeric interface of D4 domain. Additionally, as negative control or not expecting to interfere, we searched molecules preferentially docking on the apex of D1 domain. VTSAXS experiments of CD4 + molecules at ∼1:3 molar ratio showed that as expected few D4 reactive hits could retard dimerization, yet surprisingly molecules which docked at D1 domain could also derail dimerization. Additional analysis led to conclusion that there lies a systematic communication network across the structural length of sCD4 which senses binding to self and other molecules, and our work can be used to develop new (or re-purpose known) molecules as CD4-reactive immunosuppressive agents.Communicated by Ramaswamy H. Sarma.

The N-terminus region of Drp1, a Rint1 family protein is essential for cell survival and its interaction with Rad50 protein in fission yeast S.pombe.

BACKGROUND: Defects in DNA repair pathway can lead to double-strand breaks leading to genomic instability. Earlier we have shown that S.pombe Drp1, a Rint1/Tip1 family protein is required for the recovery from DNA damage. METHODS: Various truncations of Drp1 protein were constructed and their role in DNA damage response and interaction with Rad50 protein has been studied by co-immunoprecipitation and pull-down assays. RESULTS: The structural and functional analysis of Drp1 protein revealed that the N-terminus region of Drp1 is indispensable for the survival. The C-terminus truncation mutants, drp1C1Δ and drp1C2Δ exhibit temperature sensitive phenotype and are hypersensitive against DNA damaging agents with elevated level of Rad52-YFP foci at non-permissive temperature indicating the impairment for DNA damage repair pathway. The essential N-terminus region of Drp1 interacts with the C-terminus region of Rad50 and might be involved in influencing the MRN/X function. Small-angle X-ray (SAXS) analysis revealed three-domain like shapes in Drp1 protein while the C-terminus region of Rad50 exhibit unusual bulges. Computational docking studies revealed the amino acid residues at the C-terminus region of Rad50 that are involved in the interaction with the residues present at the N-terminal region of Drp1 indicating the importance of the N-terminal region of Drp1 protein. CONCLUSIONS: We have identified the region of Drp1 and Rad50 proteins that are involved in the interaction and their role in the DNA damage response pathway has been analyzed. GENERAL SIGNIFICANCE: The functional and structural aspects of fission yeast Drp1 protein and its interaction with Rad50 have been elucidated.

Biochemical characterization of ClpB protein from Mycobacterium tuberculosis and identification of its small-molecule inhibitors.

Tuberculosis, caused by pathogenic M. tuberculosis, remains a global health concern among various infectious diseases. Studies show that ClpB, a major disaggregase, protects the pathogen from various stresses encountered in the host environment. In the present study we have performed a detailed biophysical characterization of M. tuberculosis ClpB followed by a high throughput screening to identify small molecule inhibitors. The sedimentation velocity studies reveal that ClpB oligomerization varies with its concentration and presence of nucleotides. Further, using high throughput malachite green-based screening assay, we identified potential novel inhibitors of ClpB ATPase activity. The enzyme kinetics revealed that the lead molecule inhibits ClpB activity in a competitive manner. These drugs were also able to inhibit ATPase activity associated with E. coli ClpB and yeast Hsp104. The identified drugs inhibited the growth of intracellular bacteria in macrophages. Small angle X-ray scattering based modeling shows that ATP, and not its non-hydrolyzable analogs induce large scale conformational rearrangements in ClpB. Remarkably, the identified small molecules inhibited these ATP inducible conformational changes, suggesting that nucleotide induced shape changes are crucial for ClpB activity. The study broadens our understanding of M. tuberculosis chaperone machinery and provides the basis for designing more potent inhibitors against ClpB chaperone.

A novel function of Mycobacterium tuberculosis chaperonin paralog GroEL1 in copper homeostasis.

Among the two GroEL paralogs in Mycobacterium tuberculosis, GroEL1 and GroEL2, GroEL1 has a characteristic histidine-rich C terminus. Since histidine richness is likely to be involved in metal binding, we attempted to decipher the role of GroEL1 in chelating metals and the consequence on M. tuberculosis physiology. Isothermal titration calorimetry showed that GroEL1 binds copper and other metals. Mycobacterial viability assay, redox balance, and DNA protection assay concluded that GroEL1 protects from copper stress in vitro. Solution X-ray scattering and constrained modeling of GroEL1 -/+ copper ions showed reorientation of the apical domain as seen in functional assembly. We conclude that the duplication of chaperonin genes in M. tuberculosis might have led to their evolutionary divergence and consequent functional divergence of chaperonins.