Minichromosome maintenance proteins in eukaryotic chromosome segregation.

Minichromosome maintenance (Mcm) proteins are well-known for their functions in DNA replication. However, their roles in chromosome segregation are yet to be reviewed in detail. Following the discovery in 1984, a group of Mcm proteins, known as the ARS-nonspecific group consisting of Mcm13, Mcm16-19, and Mcm21-22, were characterized as bonafide kinetochore proteins and were shown to play significant roles in the kinetochore assembly and high-fidelity chromosome segregation. This review focuses on the structure, function, and evolution of this group of Mcm proteins. Our in silico analysis of the physical interactors of these proteins reveals that they share non-overlapping functions despite being copurified in biochemically stable complexes. We have discussed the contrasting results reported in the literature and experimental strategies to address them. Taken together, this review focuses on the structure-function of the ARS-nonspecific Mcm proteins and their evolutionary flexibility to maintain genome stability in various organisms.

Helical and ß-Turn Conformations in the Peptide Recognition Regions of the VIM1 PHD Finger Abrogate H3K4 Peptide Recognition.

The PHD finger-containing VARIANT IN METHYLATION/ORTHRUS (VIM/ORTH) family of proteins in Arabidopsis consists of functional homologues of mammalian UHRF1 and is required for the maintenance of DNA methylation. Comparison of the sequence with those of other PHD fingers implied that VIM1 and VIM3 PHD could recognize lysine 4 of histone H3 (H3K4) through interactions mediated by a conserved aspartic acid. However, our calorimetric and modified histone peptide array binding studies suggested that neither H3K4 nor other histone marks are recognized by VIM1 and VIM3 PHD fingers. Here, we report a 2.6 Å resolution crystal structure of the VIM1 PHD finger and demonstrate significant structural changes in the putative H3 recognition segments in contrast to canonical H3K4 binding PHD fingers. These changes include (i) the H3A1 binding region, (ii) strand ß1 that forms an intermolecular ß-sheet with the H3 peptide, and (iii) an aspartate-containing motif involved in salt bridge interaction with H3K4, which together appear to abrogate recognition of H3K4 by the VIM1 PHD finger. To understand the significance of the altered structural features in the VIM1 PHD that might prevent histone H3 recognition, we modeled a chimeric VIM1 PHD (chmVIM1 PHD) by grafting the peptide binding structural features of the BHC80 PHD onto the VIM1 PHD. Molecular dynamics simulation and metadynamics analyses revealed that the chmVIM1 PHD-H3 complex is stable and also showed a network of intermolecular interactions similar to those of the BHC80 PHD-H3 complex. Collectively, this study reveals that subtle structural changes in the peptide binding region of the VIM1 PHD abrogate histone H3 recognition.

A simple monophasic LGPY medium for routine maintenance of Leishmania donovani promastigotes.

Here, we report a simple, economic and autoclavable monophasic LGPY medium supplemented with 10% fetal bovine serum (FBS), for routine maintenance of Leishmania donovani promastigotes for laboratory use. In comparison to commercially available M199 and RPMI-1640 media, LGPY has shown approximately seven fold more cell growth. The parasite has been observed to survive in the medium for at least 15 days post-inoculation. The medium also supports long-term sub-passaging of the promastigotes and can also be stored at 4 °C or room temperature for 14 months and 45 days, respectively.

Computational characterization of substrate and product specificities, and functionality of S-adenosylmethionine binding pocket in histone lysine methyltransferases from Arabidopsis, rice and maize.

Histone lysine methylation by histone lysine methyltransferases (HKMTs) has been implicated in regulation of gene expression. While significant progress has been made to understand the roles and mechanisms of animal HKMT functions, only a few plant HKMTs are functionally characterized. To unravel histone substrate specificity, degree of methylation and catalytic activity, we analyzed Arabidopsis Trithorax-like protein (ATX), Su(var)3-9 homologs protein (SUVH), Su(var)3-9 related protein (SUVR), ATXR5, ATXR6, and E(Z) HKMTs of Arabidopsis, maize and rice through sequence and structure comparison. We show that ATXs may exhibit methyltransferase specificity toward histone 3 lysine 4 (H3K4) and might catalyse the trimethylation. Our analyses also indicate that most SUVH proteins of Arabidopsis may bind histone H3 lysine 9 (H3K9). We also predict that SUVH7, SUVH8, SUVR1, SUVR3, ZmSET20 and ZmSET22 catalyse monomethylation or dimethylation of H3K9. Except for SDG728, which may trimethylate H3K9, all SUVH paralogs in rice may catalyse monomethylation or dimethylation. ZmSET11, ZmSET31, SDG713, SDG715, and SDG726 proteins are predicted to be catalytically inactive because of an incomplete S-adenosylmethionine (SAM) binding pocket and a post-SET domain. E(Z) homologs can trimethylate H3K27 substrate, which is similar to the Enhancer of Zeste homolog 2 of humans. Our comparative sequence analyses reveal that ATXR5 and ATXR6 lack motifs/domains required for protein-protein interaction and polycomb repressive complex 2 complex formation. We propose that subtle variations of key residues at substrate or SAM binding pocket, around the catalytic pocket, or presence of pre-SET and post-SET domains in HKMTs of the aforementioned plant species lead to variations in class-specific HKMT functions and further determine their substrate specificity, the degree of methylation and catalytic activity.

Computational and in vitro binding studies of theophylline against phosphodiesterases functioning in sperm in presence and absence of pentoxifylline.

Fertility is a result of a synergy among the sperm's various functions including capacitation, motility, chemotaxis, acrosome reaction, and, finally, the fertilization of the oocyte. Subpar motility is the most common cause of infertility in males. Cyclic adenosine monophosphate (cAMP) signalling underlies motility and is depleted by the phosphodiesterases (PDEs) in sperm, such as PDE10A, PDE1, and PDE4. Therefore, the PDE inhibitor (PDEI) category of fertility drugs aim to enhance motility in assisted reproduction technologies (ARTs) through inhibition of PDEs, though they might have adverse effects on other physiological variables. For example, the popular drug pentoxifylline (PTX), widely used in ARTs, improves motility but causes premature acrosome reaction and exerts toxicity on the fertilized oocyte. Another xanthine-derived drug, theophylline (TP), has been repurposed for treating infertility, but its mechanism of PDE inhibition remains unexplored. Here, using biophysical and computational approaches, we identified that TP binds to the same binding pocket as PTX with higher affinity than PTX. We also found that PTX and TP co-bind to the same binding pocket, but at different sites.

PAR recognition by PARP1 regulates DNA-dependent activities and independently stimulates catalytic activity of PARP1.

Poly(ADP-ribosyl)ation is predominantly catalyzed by Poly(ADP-ribose) polymerase 1 (PARP1) in response to DNA damage, mediating the DNA repair process to maintain genomic integrity. Single-strand (SSB) and double-strand (DSB) DNA breaks are bona fide stimulators of PARP1 activity. However, PAR-mediated PARP1 regulation remains unexplored. Here, we report ZnF3, BRCT, and WGR, hitherto uncharacterized, as PAR reader domains of PARP1. Surprisingly, these domains recognize PARylated protein with a higher affinity compared with PAR but bind with weak or no affinity to DNA breaks as standalone domains. Conversely, ZnF1 and ZnF2 of PARP1 recognize DNA breaks but bind weakly to PAR. In addition, PAR reader domains, together, exhibit a synergy to recognize PAR or PARylated protein. Further competition-binding studies suggest that PAR binding releases DNA from PARP1, and the WGR domain facilitates DNA release. Unexpectedly, PAR showed catalytic stimulation of PARP1 but hampered the DNA-dependent stimulation. Altogether, our work discovers dedicated high-affinity PAR reader domains of PARP1 and uncovers a novel mechanism of allosteric regulation of DNA-dependent and DNA-independent activities of PARP1 by its catalytic product PAR.

Mechanistic insights into allosteric regulation of methylated DNA and histone H3 recognition by SRA and SET domains of SUVH5 and the basis for di-methylation of lysine residue.

Su-(var)3-9 homologue 5 (SUVH5), a member of SUVH family of histone lysine methyltransferase (HKMT) in Arabidopsis, is involved in epigenetic regulation of chromatin by recognizing 5-methyl-cytosine (5mC), in both CpG and non-CpG DNA context, through SRA domain and simultaneously performing the di-methylation of lysine 9 of histone H3 (H3K9) through SET domain. Here, we establish that the SET domain of SUVH5 allosterically restricts the SRA domain to the 5mC containing strand(s) of fully methylated CpG, hemi-methylated CpG and methylated CpHpH DNA. In addition, SET domain enhances the binding affinity of the SRA-SET dual domains to fully-mCpG but not to hemi-mCpG. Also, the recognition of methylated DNA by the SRA positively influences the recognition of H3K9 by the SET domain. Our further studies revealed that the SET domain recognizes the "A(R/K)KST" motif present in H3K9 and in other histone H2A variants. Further, computational analyses and quantum mechanics/molecular mechanics calculations explain the bases for robust mono-MTase but weak di-MTase activities of SUVH5. Given that the majority of eukaryotic proteins, including those involved in epigenetic gene regulation, contain more than one domain, our study suggests that understanding the allosteric regulation among multiple domains of proteins is relevant for unravelling biological outcomes.

Designing ferritin nanocage based vaccine candidates for SARS-CoV-2 by in silico engineering of its HLA I and HLA II epitope peptides.

New variants of SARS-CoV-2 are continuously being reported. To curtail the spread of this virus, it is essential to find an efficient and potent vaccine. Here, we report in silico designing of a protein (ferritin: FR) nanocage fused with multiple epitopes identified using the immuno-informatics approach and high-throughput screening. Employing computational approaches, we identified potential epitopes from membrane, nucleocapsid, and envelope proteins of SARS-CoV-2 and docked them on the selected human leukocyte antigen Class I and II receptors, then the stability of the complexes was assessed using molecular dynamics simulation studies. We have engineered chimeric ferritin nanocage, chm66FR, with the nested peptide of 10 epitopes by replacing the loop region at the 66th position of the nanocage, then its stability was confirmed using metadynamics simulation. Further, we used the homotrimeric '6-helical bundle' of the spike protein to engineer the chimeric 6HB (chm6HB). The chm6HB is, engineered with three epitope peptides, mounted on the N-terminal trimeric interface of the chm66FR to generate the chm6HB-chm66FR, which contains 15 epitope peptides. Chimeric FR nanocages and the chm6HB could be potential vaccine candidates against strains of SARS-CoV-2. These multivalent and multiple epitopes protein nanocages and scaffolds could mount both humoral and T-cell mediated immune responses against SARS-CoV-2.Communicated by Ramaswamy H. Sarma.

Regulation of PARP1 and its apoptotic variant activity by single-stranded DNA.

PARP1 is a nuclear protein involved in the maintenance of genomic stability. It catalyses the formation of poly(ADP-ribose) (PAR) to recruit repair proteins at the site of DNA lesions, such as double-strand and single-strand breaks. In the process of DNA replication or repair, there could occur stretch of ssDNA, usually protected by ssDNA binding proteins, but when present in abundance can turn into DNA beaks and cause cell death. PARP1 is an extremely sensitive sensor of DNA breaks; however, the interaction of PARP1 with single-stranded DNA (ssDNA) remains unexplored. Here, we report that the two Zn-fingers, ZnF1 and ZnF2, of PARP1, mediate high-affinity recognition of ssDNA. Our studies suggest that although PAR and ssDNA are chemical analogues, they are recognized by a distinct set of domains of PARP1, yet PAR not only induces dislodging of ssDNA from PARP1 but also hampers the ssDNA-dependent PARP1 activity. It is noteworthy that PAR carrier apoptotic fragment PARP1ΔZnF1-2 gets cleaved from PARP1 to facilitate apoptosis, leaving behind the DNA-bound ZnF1-ZnF2PARP1 . Our studies demonstrate that the PARP1ΔZnF1-2 is competent for ssDNA-dependent stimulation only in the presence of another apoptotic fragment ZnF1-ZnF2PARP1 , suggesting the indispensability of DNA-bound ZnF1-ZnF2PARP1 dual domains for the same.

Allosteric crosstalk in modular proteins: Function fine-tuning and drug design.

Modular proteins are regulatory proteins that carry out more than one function. These proteins upregulate or downregulate a biochemical cascade to establish homeostasis in cells. To switch the function or alter the efficiency (based on cellular needs), these proteins require different facilitators that bind to a site different from the catalytic (active/orthosteric) site, aka 'allosteric site', and fine-tune their function. These facilitators (or effectors) are allosteric modulators. In this Review, we have discussed the allostery, characterized them based on their mechanisms, and discussed how allostery plays an important role in the activity modulation and function fine-tuning of proteins. Recently there is an emergence in the discovery of allosteric drugs. We have also emphasized the role, significance, and future of allostery in therapeutic applications.

Structure and Cooperativity in Substrate-Enzyme Interactions: Perspectives on Enzyme Engineering and Inhibitor Design.

Enzyme-based synthetic chemistry provides a green way to synthesize industrially important chemical scaffolds and provides incomparable substrate specificity and unmatched stereo-, regio-, and chemoselective product formation. However, using biocatalysts at an industrial scale has its challenges, like their narrow substrate scope, limited stability in large-scale one-pot reactions, and low expression levels. These limitations can be overcome by engineering and fine-tuning these biocatalysts using advanced protein engineering methods. A detailed understanding of the enzyme structure and catalytic mechanism and its structure-function relationship, cooperativity in binding of substrates, and dynamics of substrate-enzyme-cofactor complexes is essential for rational enzyme engineering for a specific purpose. This Review covers all these aspects along with an in-depth categorization of various industrially and pharmaceutically crucial bisubstrate enzymes based on their reaction mechanisms and their active site and substrate/cofactor-binding site structures. As the bisubstrate enzymes constitute around 60% of the known industrially important enzymes, studying their mechanism of actions and structure-activity relationship gives significant insight into deciding the targets for protein engineering for developing industrial biocatalysts. Thus, this Review is focused on providing a comprehensive knowledge of the bisubstrate enzymes' structure, their mechanisms, and protein engineering approaches to develop them into industrial biocatalysts.

Mechanistic insights into recognition of symmetric methylated cytosines in CpG and non-CpG DNA by UHRF1 SRA.

Non-CpG DNA methylation (non-mCpG) is enriched in the genome of brain neurons and germline cells in mammals. Accumulation of non-mCpG during postnatal brain development correlates with gene regulation and inactivation of distal regulatory elements. Recently, UHRF1 has been found to contribute to de novo non-CpG methylation, however, whether UHRF1 could recognize non-mCpG is unknown. Here, we have demonstrated through calorimetric measurements that the UHRF1 SRA can recognize mCpH and fully-mCpHpG, types of non-mCpG. Our ITC binding studies endorse the preferential reading of hemi-mCpG by UHRF1 SRA and also show 6-fold weaker binding for fully-mCpG than hemi-mCpG. Despite presence of symmetrical (5-methyl cytosine) 5mCs, stoichiometry of 1:1 for UHRF1 SRA binding to fully-mCpG indicates that UHRF1 SRA may not form a stable complex with fully-mCpG DNA. Contrarily, UHRF1 SRA recognizes fully-mCpHpG with a stoichiometry of 2:1 protein to DNA duplex with binding affinity higher than fully-mCpG. Our crystal structure of UHRF1 SRA bound to fully-mCpHpG DNA reveals dual flip-out mechanism of 5mC recognition. Metadynamics studies corroborates with ITC data that UHRF1 SRA could not form a stable complex with fully-mCpG DNA. Altogether, this study demonstrates that UHRF1 SRA recognizes non-mCpG DNA and exhibits contrasting mechanisms for hemi-mCpG and fully-mCpHpG DNA recognition.

Triclosan affects motor function in zebrafish larva by inhibiting ache and syn2a genes.

The widespread use of triclosan in personal care products as an antimicrobial agent is leading to its alarming tissue-bioaccumulation including human brain. However, knowledge of its potential effects on the vertebrate nervous system is still limited. Here, we hypothesized that sublethal triclosan concentrations are potent enough to alter motor neuron structure and function in zebrafish embryos exposed for prolonged duration. In this study, zebrafish embryos were used as vertebrate-animal model. Prolonged exposure (up to 4 days) of 0.6 mg/L (LC50, 96 h) and 0.3 mg/L (

Structure-based redesigning of pentoxifylline analogs against selective phosphodiesterases to modulate sperm functional competence for assisted reproductive technologies.

Phosphodiesterase (PDE) inhibitors, such as pentoxifylline (PTX), are used as pharmacological agents to enhance sperm motility in assisted reproductive technology (ART), mainly to aid the selection of viable sperm in asthenozoospermic ejaculates and testicular spermatozoa, prior to intracytoplasmic sperm injection (ICSI). However, PTX is reported to induce premature acrosome reaction (AR) and, exert toxic effects on oocyte function and early embryo development. Additionally, in vitro binding studies as well as computational binding free energy (ΔGbind) suggest that PTX exhibits weak binding to sperm PDEs, indicating room for improvement. Aiming to reduce the adverse effects and to enhance the sperm motility, we designed and studied PTX analogues. Using structure-guided in silico approach and by considering the physico-chemical properties of the binding pocket of the PDEs, designed analogues of PTX. In silico assessments indicated that PTX analogues bind more tightly to PDEs and form stable complexes. Particularly, ex vivo evaluation of sperm treated with one of the PTX analogues (PTXm-1), showed comparable beneficial effect at much lower concentration-slower AR, higher DNA integrity and extended longevity of  spermatozoa and  superior embryo quality. PTXm-1 is proposed to be a better pharmacological agent for ART than PTX for sperm function enhancement.

In vitro studies on non-canonical DNA binding specificities of KAP6 and HMO1 and mechanistic insights into DNA bound and unbinding dynamics of KAP6.

High mobility group box (HMGB) members are DNA binding proteins with varied functions present across kingdoms. The mechanism by which HMGBs with varying number of HMG boxes are able to carry out similar functions, are poorly understood. Moreover, how non-canonical DNAs are recognized by HMGB proteins is not clear. To address these, we carried out detailed biochemical and computational studies to characterize two HMGB members- Kinetoplast associated protein (KAP6) of Trypanosoma and High mobility group protein 1 (HMO1) from yeast. Here, we report that KAP6 binds non-canonical DNAs tighter than B-form DNA. Among non-canonical DNAs, KAP6 has the highest affinity for splayed and flap structures, but least for Holliday Junction (HJ). In contrast, HMO1 binds tighter to HJ. Computational analysis show that the secondary structural elements involved in DNA interaction are conserved in HMGB members KAP6 and mitochondrial transcription factor A. Simulation analyses revealed that the ~90° bend in DNA induced by KAP6 HMG box is a result of two ~45° bends, by Helix 1 and Helix 2 of the protein. Our data also suggests that the orthologs of HMO1 and KAP6 are oligomers in solution, which could be necessary for their functioning such as DNA bending and looping.

Computational insights into mechanism of AIM4-mediated inhibition of aggregation of TDP-43 protein implicated in ALS and evidence for in vitro inhibition of liquid-liquid phase separation (LLPS) of TDP-432C-A315T by AIM4.

TDP-43 is an RNA/DNA-binding protein which is also implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS) disease. TDP-43's cytoplasmic mis-localization, liquid-liquid phase separation (LLPS) due to RNA depletion and aggregation, are proposedly important TDP-43-toxicity causing mechanisms. So far, therapeutic options for ALS are extremely ineffective hence, multi-faceted approaches such as targeting the oxidative stress and inhibiting the TDP-43's aggregation, are being actively pursued. Recently, we have identified an acridine derivative, AIM4, as an anti-TDP-43 aggregation molecule however, its mechanism is not deciphered. Here, we have utilized computational tools to examine binding site(s) of AIM4 in the TDP-43 structure and compared with other relevant compounds. We find that AIM4 has a binding site in the C-terminal amyloidogenic region (aa: 288-319), with Gly-288 & Phe-289 residues which are also important for TDP-43's LLPS. Importantly, alike to previously reported effects of RNA, AIM4 could also inhibit the in vitro LLPS of a C-terminal fragment TDP-432C bearing an A315T familial mutation. Furthermore, isothermal titration calorimetry (ITC) data also support the binding of AIM4 to TDP-432C-A315T. This antagonism of AIM4 towards TDP-43's LLPS and presence of binding site of AIM4 on TDP-43 support AIM4's potential to be an important molecule towards ALS therapeutic research.

Dynamic Basis for Auranofin Drug Recognition by Thiol-Reductases of Human Pathogens and Intermediate Coordinated Adduct Formation with Catalytic Cysteine Residues.

In all the living systems, reactive oxygen species (ROS) metabolism provides resistance against internal and external oxidative stresses. Auranofin (AF), an FDA-approved gold [Au(I)]-conjugated drug, is known to selectively target thiol-reductases, key enzymes involved in ROS metabolism. AF has been successfully tested for its inhibitory activity through biochemical studies, both in vitro and in vivo, against a diverse range of pathogens including protozoa, nematodes, bacteria, and so forth. Cocrystal structures of thiol-reductases complexed with AF revealed that Au(I) was coordinately linked to catalytic cysteines, but the mechanism of transfer of Au(I) from AF to catalytic cysteines still remains unknown. In this study, we have employed computational approaches to understand the interaction of AF with thiol-reductases of selected human pathogens. A similar network of interactions of AF was observed in all the studied enzymes. Also, we have shown that tailor-made analogues of AF can be designed against selective thiol-reductases for targeted inhibition. Molecular dynamics studies show that the AF-intermediates, tetraacetylthioglucose (TAG)-gold, and triethylphosphine (TP)-gold, coordinately linked to one of catalytic cysteines, remain stable in the binding pocket of thiol-reductases for Leishmania infantum and Plasmodium falciparum (PfTrxR). This suggests that the TP and TAG moieties of AF may be sequentially eliminated during the transfer of Au(I) to catalytic cysteines of the receptor.

Biochemical and dynamic basis for combinatorial recognition of H3R2K9me2 by dual domains of UHRF1.

UHRF1 is a multi-domain protein comprising of a tandem tudor (UHRF1 TTD), a PHD finger, and a SET and RING-associated domain. It is required for the maintenance of CG methylation, heterochromatin formation and DNA repair. Isothermal titration calorimetry binding studies of unmodified and methylated lysine histone peptides establish that the UHRF1 TTD binds dimethylated Lys9 on histone H3 (H3K9me2). Further, MD simulation and binding studies reveal that TTD-PHD of UHRF1 (UHRF1 TTD-PHD) preferentially recognizes dimethyl-lysine status. Importantly, we show that Asp145 in the binding pocket determines the preferential recognition of the dimethyl-ammonium group of H3K9me2. Interestingly, PHD finger of the UHRF1 TTD-PHD has a negligible contribution to the binding affinity for recognition of K9me2 by the UHRF1 TTD. Surprisingly, Lys4 methylation on H3 peptide has an insignificant effect on combinatorial recognition of R2 and K9me2 on H3 by the UHRF1 TTD-PHD. We propose that subtle variations of key residues at the binding pocket determine status specific recognition of histone methyl-lysines by the reader domains.