G protein-coupled receptors (GPCRs) are the largest group of receptors involved in various types of signaling. GPCR signaling is regulated via receptor phosphorylation by G protein-coupled receptor kinases 5 (GRK5). Calmodulin (CaM), a universal Ca2+ sensor, inhibits receptor phosphorylation by binding to GRK5. However, the inhibitor malbrancheamide (MBC), which binds at CaM C-lobe, allows for receptor phosphorylation. To understand the phosphorylation mechanism by GRK5, we carried out a MD simulation of the CaM/GRK5 complex in the presence and absence of the MBC inhibitor. The lipid binding domain (LBD) of GRK5 adopted different positions in the presence and absence of inhibitor. Furthermore, the inhibitor MBC restricted the movement of the N-lobe tether (NLT) loop, probably blocking the autophosphorylation of GRK5.

Protein-protein interaction (PPI) is a crucial event for many biological functions. Studying the molecular details of PPI requires structure determination using X-ray crystallography, nuclear magnetic resistance (NMR), and single particle Cryo-EM. However, sometimes it is not easy to solve the complex structure for various reasons. For example, complex may be unstable, not enough protein expression for structural studies, etc. Further, PPI are intricate processes, and its molecular details cannot be fully explained by experimental observations. Here, we describe a quick and simple method to study the PPI using the combinatorial approach of molecular dynamics simulation and biophysical methods.

Cervical cancer is the fourth leading cause of death in women worldwide, with 99% of cases associated with a human papillomavirus (HPV) infection. Given that HPV prophylactic vaccines do not exert a therapeutic effect in individuals previously infected, have low coverage of all HPV types, and have poor accessibility in developing countries, it is unlikely that HPV-associated cancers will be eradicated in the coming years. Therefore, there is an emerging need for the development of anti-HPV drugs. Considering HPV E6's oncogenic role, this protein has been proposed as a relevant target for cancer treatment. In the present work, we employed in silico tools to discover potential E6 inhibitors, as well as biochemical and cellular assays to understand the action of selected compounds in HPV-positive cells (Caski and HeLa) vs. HPV-negative (C33A) and non-carcinogenic (NHEK) cell lines. In fact, by molecular docking and molecular dynamics simulations, we found three phenolic compounds able to dock in the E6AP binding pocket of the E6 protein. In particular, lucidin and taxifolin were able to inhibit E6-mediated p53 degradation, selectively reduce the viability, and induce apoptosis in HPV-positive cells. Altogether, our data can be relevant for discovering promising leads for the development of specific anti-HPV drugs.

The disease COVID-19 has caused heavy socio-economic burden and there is immediate need to control it. The disease is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus. The viral entry into human cell depends on the attachment of spike (S) protein via its receptor binding domain (RBD) to human cell receptor angiotensin-converting enzyme 2 (hACE2). Thus, blocking the virus attachment to hACE2 could serve as potential therapeutics for viral infection. We have designed a peptide inhibitor (ΔABP-α2) targeting the RBD of S protein using in-silico approach. Docking studies and computed affinities suggested that peptide inhibitor binds at the RBD with ∼95-fold higher affinity than hACE2. Molecular dynamics (MD) simulation confirms the stable binding of inhibitor to hACE2. Immunoinformatics studies suggest non-immunogenic and non-toxic nature of peptide. Thus, the proposed peptide could serve as potential blocker for viral attachment. Communicated by Ramaswamy H. Sarma

Calmodulin, a ubiquitous eukaryotic calcium sensor responsible for the regulation of many fundamental cellular processes, is a highly flexible protein and exhibits an unusually wide range of conformations. Furthermore, CaM is known to interact with more than 300 cellular targets. Molecular dynamics (MD) simulation trajectories suggest that EF-hand loops show different magnitudes of flexibility. Therefore, the four EF-hand motifs have different affinities for Ca2+ ions, which enables CaM to function on wide range of Ca2+ ion concentrations. EF-hand loops are 2-3 times more flexible in apo CaM whereas least flexible in Ca2+/CaM-IQ motif complexes. We report a unique intermediate conformation of Ca2+/CaM while transitioning from extended to compact form. We also report the complex formation process between Ca2+/CaM and IQ CaM-binding motifs. Our results showed how IQ motif recognise its binding site on the CaM and how CaM transforms from extended to compact form upon binding to IQ motif.

The SARS-CoV-2 virus has caused a pandemic and is public health emergency of international concern. As of now, no registered therapies are available for treatment of coronavirus infection. The viral infection depends on the attachment of spike (S) glycoprotein to human cell receptor angiotensin-converting enzyme 2 (ACE2). We have designed a protein inhibitor (ΔABP-D25Y) targeting S protein using computational approach. The inhibitor consists of two α helical peptides homologues to protease domain (PD) of ACE2. Docking studies and molecular dynamic simulation revealed that the inhibitor binds exclusively at the ACE2 binding site of S protein. The computed binding affinity of the inhibitor is higher than the ACE2 and thus will likely out compete ACE2 for binding to S protein. Hence, the proposed inhibitor ΔABP-D25Y could be a potential blocker of S protein and receptor binding domain (RBD) attachment.

Members of the ATP-binding cassette F (ABC-F) proteins confer resistance to several classes of clinically important antibiotics through ribosome protection. Recent structures of two ABC-F proteins, Pseudomonas aeruginosa MsrE and Bacillus subtilis VmlR bound to ribosome have shed light onto the ribosome protection mechanism whereby drug resistance is mediated by the antibiotic resistance domain (ARD) connecting the two ATP binding domains. ARD of the E site bound MsrE and VmlR extends toward the drug binding region within the peptidyl transferase center (PTC) and leads to conformational changes in the P site tRNA acceptor stem, the PTC, and the drug binding site causing the release of corresponding drugs. The structural similarities and differences of the MsrE and VmlR structures likely highlight an universal ribosome protection mechanism employed by antibiotic resistance (ARE) ABC-F proteins. The variable ARD domains enable this family of proteins to adapt the protection mechanism for several classes of ribosome-targeting drugs. ARE ABC-F genes have been found in numerous pathogen genomes and multi-drug resistance conferring plasmids. Collectively they mediate resistance to a broader range of antimicrobial agents than any other group of resistance proteins and play a major role in clinically significant drug resistance in pathogenic bacteria. Here, we review the recent structural and biochemical findings on these emerging resistance proteins, offering an update of the molecular basis and implications for overcoming ABC-F conferred drug resistance.

Dunaliella has been extensively studied due to its intriguing adaptation to high salinity. Its di-domain glycerol-3-phosphate dehydrogenase (GPDH) isoform is likely to underlie the rapid production of the osmoprotectant glycerol. Here, we report the structure of the chimeric Dunaliella salina GPDH (DsGPDH) protein featuring a phosphoserine phosphatase-like domain fused to the canonical glycerol-3-phosphate (G3P) dehydrogenase domain. Biochemical assays confirm that DsGPDH can convert dihydroxyacetone phosphate (DHAP) directly to glycerol, whereas a separate phosphatase protein is required for this conversion process in most organisms. The structure of DsGPDH in complex with its substrate DHAP and co-factor nicotinamide adenine dinucleotide (NAD) allows the identification of the residues that form the active sites. Furthermore, the structure reveals an intriguing homotetramer form that likely contributes to the rapid biosynthesis of glycerol.

The ribosome is one of the richest targets for antibiotics. Unfortunately, antibiotic resistance is an urgent issue in clinical practice. Several ATP-binding cassette family proteins confer resistance to ribosome-targeting antibiotics through a yet unknown mechanism. Among them, MsrE has been implicated in macrolide resistance. Here, we report the cryo-EM structure of ATP form MsrE bound to the ribosome. Unlike previously characterized ribosomal protection proteins, MsrE is shown to bind to ribosomal exit site. Our structure reveals that the domain linker forms a unique needle-like arrangement with two crossed helices connected by an extended loop projecting into the peptidyl-transferase center and the nascent peptide exit tunnel, where numerous antibiotics bind. In combination with biochemical assays, our structure provides insight into how MsrE binding leads to conformational changes, which results in the release of the drug. This mechanism appears to be universal for the ABC-F type ribosome protection proteins.

Elongation factor 4 (EF4) is a member of the family of ribosome-dependent translational GTPase factors, along with elongation factor G and BPI-inducible protein A. Although EF4 is highly conserved in bacterial, mitochondrial, and chloroplast genomes, its exact biological function remains controversial.Here we present the cryo-EM reconstitution of the GTP form of EF4 bound to the ribosome with P and E site tRNAs at 3.8-Å resolution.Interestingly, our structure reveals an unrotated ribosome rather than a clockwise-rotated ribosome, as observed in the presence of EF4-GDP andPsite tRNA.In addition, we also observed a counterclockwise-rotated form of the above complex at 5.7-Å resolution.Taken together, our results shed light on the interactions formed between EF4, the ribosome, and the P site tRNA and illuminate the GTPase activation mechanism at previously unresolved detail.

EF-G, EF4, and BipA are members of the translation factor family of GTPases with a common ribosome binding mode and GTPase activation mechanism. However, topological variations of shared as well as unique domains ensure different roles played by these proteins during translation. Recent X-ray crystallography and cryo-electron microscopy studies have revealed the structural basis for the involvement of EF-G domain IV in securing the movement of tRNAs and mRNA during translocation as well as revealing how the unique C-terminal domains of EF4 and BipA interact with the ribosome and tRNAs contributing to the regulation of translation under certain conditions. EF-G, EF-4, and BipA are intriguing examples of structural variations on a common theme that results in diverse behavior and function. Structural studies of translational GTPase factors have been greatly facilitated by the use of antibiotics, which have revealed their mechanism of action.

Protein-protein interactions are key events controlling several biological processes. We have developed and employed a method to trap transiently interacting protein complexes for structural studies using glycine-rich linkers to fuse interacting partners, one of which is unstructured. Initial steps involve isothermal titration calorimetry to identify the minimum binding region of the unstructured protein in its interaction with its stable binding partner. This is followed by computational analysis to identify the approximate site of the interaction and to design an appropriate linker length. Subsequently, fused constructs are generated and characterized using size exclusion chromatography and dynamic light scattering experiments. The structure of the chimeric protein is then solved by crystallization, and validated both in vitro and in vivo by substituting key interacting residues of the full length, unlinked proteins with alanine. This protocol offers the opportunity to study crucial and currently unattainable transient protein interactions involved in various biological processes.

Elongation factor 4 (EF4) is a member of the family of ribosome-dependent translational GTPase factors, along with elongation factor G and BPI-inducible protein A. Although EF4 is highly conserved in bacterial, mitochondrial, and chloroplast genomes, its exact biological function remains controversial. Here we present the cryo-EM reconstitution of the GTP form of EF4 bound to the ribosome with P and E site tRNAs at 3.8-Å resolution. Interestingly, our structure reveals an unrotated ribosome rather than a clockwise-rotated ribosome, as observed in the presence of EF4-GDP and P site tRNA. In addition, we also observed a counterclockwise-rotated form of the above complex at 5.7-Å resolution. Taken together, our results shed light on the interactions formed between EF4, the ribosome, and the P site tRNA and illuminate the GTPase activation mechanism at previously unresolved detail.

BPI-inducible protein A (BipA) is a member of the family of ribosome-dependent translational GTPase (trGTPase) factors along with elongation factors G and 4 (EF-G and EF4). Despite being highly conserved in bacteria and playing a critical role in coordinating cellular responses to environmental changes, its structures (isolated and ribosome bound) remain elusive. Here, we present the crystal structures of apo form and GTP analog, GDP, and guanosine-3',5'-bisdiphosphate (ppGpp)-bound BipA. In addition to having a distinctive domain arrangement, the C-terminal domain of BipA has a unique fold. Furthermore, we report the cryo-electron microscopy structure of BipA bound to the ribosome in its active GTP form and elucidate the unique structural attributes of BipA interactions with the ribosome and A-site tRNA in the light of its possible function in regulating translation.

Summary A systematic review of eight ulnar fractures in seven patients with bisphosphonate therapy was performed to describe the characteristics and predisposing factors. The proximal ulna is likely to be fractured, especially in the dominant limb of elderly female patients using walking aids after 7 to 15 years of bisphosphonate use. Introduction Long-term bisphosphonate use has been suggested to result in decreased bone remodelling and increased risk of atypical fractures. While the relationship between bisphosphonate use and atypical femoral fractures has been extensively studied, there is relative rarity and unawareness of these fractures in the forearm. We conducted a systematic review of existing case reports to better describe the characteristics and predisposing factors for fractures occurring in patients with bisphosphonate therapy. Methods The systematic review was conducted according to PRISMA guidelines. All studies with ulnar fractures in individuals with history of bisphosphonate use were included, with data extracted and analysed in totality. Results Seven patients with eight fractures are included. Predisposing factors include elderly females requiring walking aids. There is a propensity for the proximal ulna to be fractured, especially in the dominant limb used for ambulation or transfer. All patients were on bisphosphonate for 7 to 15 years. All fractures were atraumatic, non-comminuted, transverse in configuration, had localised periosteal or endosteal thickening at the fracture site and generalised cortical thickening of the diaphysis. Conclusion Ulnar fractures in patients with bisphosphonate therapy demonstrate features similar to those described for atypical femoral fractures, suggesting that these fractures could also possibly be due to bisphosphonate use. However, the ulna appears to be able to tolerate longer periods of alendronate use prior to fracture development. The mechanism and characteristics of these fractures additionally suggest the presence of repetitive stress that accumulates over time due to suppressed bone remodelling in patients on bisphosphonates, eventually resulting in these fractures.

Nat. Struct. Mol. Biol. 20, 1077–1084 (2013); published online 4 August 2013; corrected after print 12 January 2015 In view of the differences between our findings reported in this study and in Tourigny et al. (Science 340, 1235490, 2013), we have rerefined the structure with Refmac (Murshudov, G.N.et al.

Pseudomonas aeruginosa relies on cell motility and ability to form biofilms to establish infections, however, the mechanism of regulation remains obscure. Here we report that BswR, a XRE (Xenobiotic Response Element) type transcriptional regulator, plays a critical role in regulation of bacterial motility and biofilm formation in P. aeruginosa. Transcriptomic and biochemical analyses showed that BswR counteracts the repressor activity of MvaT, controls the transcription of small RNA rsmZ and regulates the biogenesis of bacterial flagella. The crystal structure of BswR was determined at 2.3 Å resolution; the monomer comprises a DNA-binding domain with a helix-turn-helix (HTH) motif in the N terminus and two helices (6 and 7) with a V-shaped arrangement in the C-terminus. In addition to the contacts between the parallel helices α5 of two monomers, the two helical extensions (6 and 7) intertwine together to form a homodimer, which is the biological function unit. Based on the result of DNase I protection assay together with structural analysis BswR homodimer, we proposed a BswR-DNA model, which suggests a molecular mechanism with which BswR could interact with DNA. Taken together, our results unveiled a novel regulatory mechanism, in which BswR controls the motility and biofilm formation of P. aeruginosa by modulating the transcription of small RNA rsmZ.

Pseudomonas aeruginosa relies on cell motility and ability to form biofilms to establish infections; however, the mechanism of regulation remains obscure. Here we report that BswR, a xenobiotic response element-type transcriptional regulator, plays a critical role in regulation of bacterial motility and biofilm formation in P. aeruginosa. Transcriptomic and biochemical analyses showed that BswR counteracts the repressor activity of MvaT, controls the transcription of small RNA rsmZ and regulates the biogenesis of bacterial flagella. The crystal structure of BswR was determined at 2.3 Å resolution; the monomer comprises a DNA-binding domain with a helix-turn-helix motif in the N terminus and two helices (α6 and α7) with a V-shaped arrangement in the C-terminus. In addition to the contacts between the parallel helices α5 of two monomers, the two helical extensions (α6 and α7) intertwine together to form a homodimer, which is the biological function unit. Based on the result of DNase I protection assay together with structural analysis of BswR homodimer, we proposed a BswR–DNA model, which suggests a molecular mechanism with which BswR could interact with DNA. Taken together, our results unveiled a novel regulatory mechanism, in which BswR controls the motility and biofilm formation of P. aeruginosa by modulating the transcription of small RNA rsmZ.

Neuromodulin (Nm) and neurogranin (Ng) are neuron-specific substrates of protein kinase C (PKC). Their interactions with Calmodulin (CaM) are crucial for learning and memory formation in neurons. Here, we report the structure of IQ peptides (24aa) of Nm/Ng complexed with CaM and their functional studies with full-length proteins. Nm/Ng and their respective IQ peptides are intrinsically unstructured; however, upon binding with CaM, IQ motifs adopt a helical conformation. Ser41 (Ser36) of Nm (Ng) is located in a negatively charged pocket in the apo CaM and, when phosphorylated, it will repel Nm/Ng from CaM. These observations explain the mechanism by which PKC-induced Ser phosphorylation blocks the association of Nm/Ng with CaM and interrupts several learning- and memory-associated functions. Moreover, the present study identified Arg as a key CaM interacting residue from Nm/Ng. This residue is crucial for CaM-mediated function, as evidenced by the inability of the Ng mutant (Arg-to-Ala) to potentiate synaptic transmission in CA1 hippocampal neurons.

Calmodulin (CaM) is a highly conserved eukaryotic protein that binds specifically to more than 100 target proteins in response to calcium (Ca(2+)) signal. CaM adopts a considerable degree of structural plasticity to accomplish this physiological role; however, the nature and extent of this plasticity remain to be fully understood. Here, we report the crystal structure of a novel trans conformation of ligand-free CaM where the relative disposition of two lobes of CaM is different, a conformation to-date not reported. While no major structural changes were observed in the independent N- and C-lobes as compared with previously reported structures of Ca(2+)/CaM, the central helix was tilted by ~90° at Arg75. This is the first crystal structure of CaM to show a drastic conformational change in the central helix, and reveals one of several possible conformations of CaM to engage with its binding partner.