Biology subjects

Borbat, P. P.

Publications and source records attributed to Borbat, P. P..

7 recordsLinked to original sources

bioRxiv2026

Affinity and Conformations of Ca2+-Calmodulin Bound to Peptides Containing the Calmodulin-Binding Motif of HIV-1 Matrix Protein

Background: Protein-protein interactions enable viruses to adapt and replicate by modifying host factors. Thermodynamic characterization of viral-host complexes can reveal viral mechanisms, broader principles of protein interactions, and properties relevant to anti-HIV-1 inhibitor development. Here, we examine the binding of the HIV-1 matrix (MA) protein to host calmodulin (CaM). MA, the N-terminal domain of Gag (group-specific antigen), directs Gag to the plasma membrane for viral assembly and maturation and may recruit CaM to facilitate this trafficking. However, the MA-CaM interaction remains poorly characterized. MA contains a noncanonical N-terminal CaM-binding motif with hydrophobic regions that may mediate CaM binding and a central, positively charged KKK cluster. Objective: We aimed to determine the binding affinities of MA to calcium-bound CaM (Ca2+-CaM), elucidate protein regions that are critical to the heterocomplex formation, and monitor the conformational alterations in Ca2+-CaM upon binding to MA. Methods: We studied Ca2+-CaM binding to synthetic MA peptides representing full-length wild type (FL WT), a C-terminal truncation lacking the KKK cluster, and an FL K26N/K27H/K28Q triple mutant of the MA CaM-binding motif. The peptides were C-terminally labeled with Cy3, and FL CaM S39C with Cy5. Binding affinity was measured by ensemble Forster resonance energy transfer (eFRET) using 100 nM Cy3-MA and increasing Cy5-Ca2+-CaM concentrations in the range of 50 nM to 2.5 microM. Conformational changes in doubly spin-labeled Ca2+-CaM upon MA binding were probed by double electron-electron resonance (DEER) spectroscopy. Results: Cy3 quenching and FRET analyses of complexes with Cy5-Ca2+-CaM yielded dissociation constants of ~80 nM for FL WT MA, ~65 nM for the truncation, and ~250 nM for the triple mutant, with corresponding binding free energies of ~9.8 to ~9 kcal/mol. These values indicate stable, specific complexes and show that the KKK cluster contributes little to MA-Ca2+-CaM binding. DEER measurements of Ca2+-CaM labeled at S39C and A103C showed that FL WT MA binding shifted the ~5.3 nm interspin distance of extended Ca2+-CaM to a broader ~1.8-3 nm distribution, indicating a major CaM restructuring. Conclusion: These findings define key energetic and structural features of MA-Ca2+-CaM interactions and clarify how HIV-1 proteins exploit CaM.

biophysics↗

bioRxiv2025

The soluble HIV-1 Vpu protein interacts with calmodulin in a Ca2+-dependent manner

The HIV-1 Vpu membrane protein is crucial to the virus lifecycle. Our recent studies revealed soluble Vpu oligomers, prompting further investigation into their interactions with cellular proteins. Notably, Vpu may form a complex with calmodulin (CaM) due to its putative CaM-binding motif; however, experimental proof of this association remained unavailable. Here, we present definitive experimental evidence that the soluble Vpu complex interacts in vitro with calcium-bound CaM (Ca2+-CaM) its active form. Using double electron electron-resonance (DEER) spectroscopy and protein spin labeling, we detected the formation of a soluble Vpu-Ca2+-CaM complex. Both the full-length (FL) and truncated C-terminal regions of Vpu bind Ca2+-CaM. DEER experiments on a spin-labeled CaM cysteine mutant S39C/A103C revealed that, upon association with Vpu, Ca2+-CaM undergoes a transition from an open to a more closed conformation, consistent with previous reports of Ca2+-CaM interactions with other proteins. Furthermore, we observed that the binding of Vpu to Ca2+-CaM leads to dissociation of soluble Vpu oligomers, as evidenced by a reduction in DEER modulation depth for FL Vpu spin-labeled at residue L42C. FRET analysis with a fluorescently labeled C-terminal cysteine mutant of Vpu confirmed this result. Like FL Vpu, the Vpu C-terminal region forms soluble homooligomers that dissociate upon binding to Ca2+-CaM. Collectively, our results suggest that soluble Vpu and Ca2+-CaM form an equimolar complex. DEER analysis of Vpu C-terminal region spin-labeled at residues Q36C/I61C demonstrated that Vpu undergoes significant conformational changes to facilitate Ca2+-CaM binding. These findings could be relevant to Vpu-CaM interactions under physiological conditions.

biophysics↗

bioRxiv2024

A comparative study of influenza A M2 protein conformations in DOPC/DOPS liposomes and in native E. coli membranes

We compared the conformations of the transmembrane domain (TMD) of influenza A M2 (IAM2) protein reconstituted at pH 7.4 in DOPC/DOPS bilayers to those in isolated E. coli membranes, having preserved its native proteins and lipids. IAM2 is a single-pass transmembrane protein known to assemble into homo-tetrameric proton channel. To represent this channel, we made a construct containing the IAM2s TMD region flanked by the juxtamembrane residues. The single cysteine substitute, L43C, of leucine located in the bilayer polar region was paramagnetically tagged with a methanethiosulfonate nitroxide label for the ESR (electron spin resonance) study. We compared the conformations of the spin-labeled IAM2 residing in DOPC/DOPS and native E. coli membranes using continuous-wave (CW) ESR and double electron-electron resonance (DEER) spectroscopy. The total protein-to-lipid molar ratio spanned the range from 1:230 to 1:10,400. The CW ESR spectra corresponded to a nearly rigid limit spin label dynamics in both environments. In all cases, the DEER data were reconstructed into the distance distributions showing well-resolved peaks at 1.68 nm and 2.37 nm. The peak distance ratio was 1.41{+/-}0.2 and the amplitude ratio was 2:1. This is what one expects from four nitroxide spin-labels located at the corners of a square, indicative of an axially symmetric tetramer. Distance modeling of DEER data with molecular modeling software applied to the NMR molecular structures (PDB: 2L0J) confirmed the symmetry and closed state of the C-terminal exit pore of the IAM2 tetramer in agreement with the NMR model. Thus, we can conclude that IAM2 TMD has similar conformations in model and native E. coli membranes of comparable thickness and fluidity, notwithstanding the complexity of the E. coli membranes caused by their lipid diversity and the abundance of integral and peripheral membrane proteins.

biophysics↗

bioRxiv2023

The crystal structure of bacteriophage lambda RexA provides novel insights into the DNA binding properties of Rex-like phage exclusion proteins

RexA and RexB function as an exclusion system that prevents bacteriophage T4rII mutants from growing on E. coli {lambda} phage lysogens. Recent data established that RexA is a non-specific DNA binding protein that can act independently of RexB to bias the {lambda} bistable switch toward the lytic state, preventing conversion back to lysogeny. The molecular interactions underlying these activities are unknown, owing in part to a dearth of structural information. Here we present the 2.05-[A] crystal structure of the {lambda} RexA dimer, which reveals a two-domain architecture with unexpected structural homology to the recombination-associated protein RdgC. Modelling suggests that our structure adopts a closed conformation and would require significant domain rearrangements to facilitate DNA binding. Mutagenesis coupled with electromobility shift assays, limited proteolysis, and double electron-electron spin resonance spectroscopy support a DNA-dependent conformational change. In vivo phenotypes of RexA mutants suggest that DNA binding is not a strict requirement for phage exclusion but may directly contribute to modulation of the bistable switch. We further demonstrate that RexA homologs from other temperate phages also dimerize and bind DNA in vitro. Collectively, these findings advance our mechanistic understanding of Rex functions and provide new evolutionary insights into different aspects of phage biology.

microbiology↗

bioRxiv2023

HIV-1 Vpu protein forms stable oligomers in aqueous solution via its transmembrane domain self-association

We report our findings on the assembly of the HIV-1 protein Vpu into soluble oligomers. Vpu is a key to HIV-1 protein. It has been considered exclusively a single-pass membrane protein. However, we revealed that this protein forms stable oligomers in aqueous solution, which is an interesting and rather unique observation, as the number of proteins transitioning between soluble and membrane embedded states is limited. Therefore, we undertook a study to characterize these oligomers by utilizing protein engineering, size exclusion chromatography, cryoEM and electron paramagnetic resonance (EPR) spectroscopy. We found that Vpu oligomerizes via its N-terminal transmembrane domain (TM). CryoEM analyses suggest that the oligomeric state most likely is a hexamer or hexamer-to-heptamer equilibrium. Both cryoEM and EPR suggest that, within the oligomer, the distant C-terminal region of Vpu is highly flexible. To the best of our knowledge, this is the first comprehensive study on soluble Vpu. We propose that these oligomers are stabilized via possibly hydrophobic interactions between Vpu TMs. Our findings contribute valuable information about this protein properties and about protein supramolecular complexes formation. The acquired knowledge could be further used in protein engineering, and could also help to uncover possible physiological function of these Vpu oligomers.

biophysics↗

bioRxiv2022

Structure and dynamics of human perilipin 3 membrane association

Lipid droplets (LDs) are dynamic organelles that contain an oil core mainly composed of triglycerides (TAG) that is surrounded by a phospholipid monolayer and LD-associated proteins called perilipins (PLINs). During LD biogenesis, perilipin 3 (PLIN3) is recruited to nascent LDs as they emerge from the endoplasmic reticulum. Here, we analyzed how lipid composition affects PLIN3 recruitment to membrane bilayers and LDs, and the structural changes that occur upon membrane binding. We found the TAG precursors phosphatidic acid and diacylglycerol (DAG) recruit PLIN3 to membrane bilayers and define an expanded Perilipin-ADRP-Tip47 (PAT) domain that preferentially binds DAG enriched membranes. Membrane binding induces a disorder/order transition of alpha helices within the PAT domain and 11-mer repeats, with intramolecular distance measurements consistent with the expanded PAT domain adopting a folded but dynamic structure upon membrane binding. In cells, PLIN3 is recruited to DAG enriched ER membranes, and this requires both the PAT domain and 11-mer repeats. This provides molecular details of PLIN3 recruitment to nascent LDs and identifies a function of the PAT domain of PLIN3 in DAG binding.

biochemistry↗

bioRxiv2020

Engineered chemotaxis core signaling units indicate a constrained kinase-off state

Bacterial chemoreceptors, the CheA histidine kinase, and the coupling protein CheW comprise transmembrane molecular arrays with remarkable sensing properties. An unanswered question concerns how receptors turn off CheA kinase activity. Chemoreceptor cytoplasmic regions engineered to assume a trimer-of-receptor-dimers configuration form well-defined complexes with CheA and CheW and promote a kinase-off state. These mimics of core signaling units were assembled to homogeneity and investigated by site-directed spin-labeling with pulse-dipolar ESR spectroscopy (PDS), small-angle x-ray scattering, targeted protein cross-linking, and cryo-electron microscopy. The kinase-off state is especially stable, has relatively low domain mobility and associates the histidine substrate domain P1 and docking domain P2 with the kinase core. Distances measured between spin-labeled ADP molecules bound to the P4 kinase domain provide evidence for a "dipped conformation" that has been previously proposed from molecular dynamics simulations. Taken together, the data provide an experimentally restrained model for the inhibited state of the core-signaling unit and suggest that chemoreceptors indirectly sequester the kinase and substrate domains to limit histidine autophosphorylation.

biochemistry↗