Biology subjects

Polenova, T.

Publications and source records attributed to Polenova, T..

7 recordsLinked to original sources

bioRxiv2026

Structural Basis of Second-Generation HIV-1 Maturation Inhibitor Activity by Magic Angle Spinning NMR

The CA-SP1 junction of HIV-1 Gag forms a six-helix bundle (6HB) within the immature Gag lattice, sequestering the CA-SP1 cleavage site. Maturation inhibitors (MIs), such as Bevirimat (BVM) and its analogs, are an emerging class of HIV-1 antiviral compounds that stabilize Gag maturation intermediates by interfering with CA-SP1 proteolytic processing. Here, we report the binding modes of a potent second-generation BVM analog with an extended scaffold, HRF-4467, in crystalline CACTD-SP1-NC11 assemblies with inositol hexakisphosphate (IP6), as determined by magic-angle-spinning solid-state NMR (MAS NMR) spectroscopy. The atomic-level structure reveals that HRF-4467 binds asymmetrically in the SP1-NC11 six-helix bundle and stabilizes its helical conformation in the immature lattice, in both the wild-type and the BVM-resistant SP1-V7A Gag variant. Additionally, in the presence of HRF-4467, IP6 adopts distinct conformations in the wild-type CACTD-SP1-NC11 and the SP1-V7A variant. Taken together, our results establish a molecular basis for HRF-4467's improved antiviral activity relative to BVM and suggest strategies for designing inhibitors with enhanced performance against MI-resistant HIV-1 variants for potential therapeutic use.

biophysics↗

bioRxiv2025

Structure of the Disulfide-rich Modules of a Striking Tandem Repeat Protein, Avian Cysteine-Rich Eggshell Membrane Protein

Avian eggshell membrane (ESM) is fabricated within the isthmus region of the oviduct and is comprised of three juxtaposed, predominantly proteinaceous layers lying between egg white and the calcified shell. The limiting membrane is less than 0.5 {micro}m in thickness and forms the osmotic barrier for the egg. This first layer provides the foundation for the successive deposition of two mats of protein fibers. Fibers from both inner and outer layers appear to have similar amino acid compositions and are notably disulfide-rich (comprising about 10% Cys). ESM has been utilized in a wide variety of applications, including nutraceutical supplements, tissue engineering, and nanofabrication, and yet fundamental questions concerning protein composition, fiber structure, and membrane assembly remain to be resolved. We previously identified an abundant disulfide-rich structural protein in chicken ESM fibers (Cysteine-rich eggshell membrane protein; CREMP) that contains multiple tandemly repeated modules. In this work, we determine a structural model for four consecutive 2-disulfide containing CREMP modules using a variety of two- and three-dimensional solution NMR experiments. CREMP modules feature an N-terminal loop region positioned above a small beta hairpin that is stabilized by a conserved pattern of disulfide bridges between Cys1-3 and Cys2-4. While the individual CREMP modules are highly ordered, the lack of long-range inter-module restraints suggests an extended structure connected by flexible linkers. Finally, the structural information obtained in this work is considered in the context of full-length CREMP proteins and compared to two other structural proteins that contain multiple tandem repeats of 2-disulfide modules. Statement of Importance and ImpactUnder the shell of an avian egg is a tough, paper-thin, three-layered protein-based structure called the eggshell membrane. This membrane has a range of medical, biomaterial and analytical applications, and yet fundamental questions concerning protein composition and assembly remain unresolved. Here, we address the 3-dimensional structure of part of a major membrane component, cysteine-rich eggshell membrane protein. The work provides new insight into the eggshell membrane and suggests an additional avenue for de-novo protein design.

biochemistry↗

bioRxiv2025

Pushing Sensitivity and Specificity Limits in Native Structural Biology: 19F Multinuclear Dynamic Nuclear Polarization with Magic Angle Spinning

Understanding protein structures and their interactions within natural cellular environments is essential for deciphering cellular processes and advancing therapeutic development. Obtaining atomic-level information about protein structural changes in cellular contexts poses a significant challenge. Here, we introduce a 19F-based, 1H-assisted dynamic nuclear polarization (DNP) magic angle spinning (MAS) NMR approach that offers exceptionally high sensitivity and specificity, enabling background-free detection of target proteins in mammalian cells for atomic-level structural analysis. We demonstrate this methodology in A2780 cells for the human Cyclophilin A (CypA) protein with a single fluorine atom incorporated in the sole tryptophan residue. We achieved significant sensitivity gains through 1H-19F cross-polarization (CP), with subsequent 19F-13C double CP providing unique structural information. Remarkably, using 1H-19F13C magnetization transfer allowed selective detection of 13C signals from CypA residues up to 6 [A] away from the fluorine label. Taken together, our study establishes a framework for investigating protein structure, dynamics, and interactions in mammalian cells by DNP MAS NMR.

biophysics↗

bioRxiv2025

Structural Basis for HIV-1 Maturation Inhibition by PF-46396 Determined by MAS NMR

Among the different types of HIV-1 maturation inhibitors, those that stabilize the junction between the capsid protein C-terminal domain (CACTD) and the spacer peptide 1 (SP1) within the immature Gag lattice are promising candidates for antiretroviral therapies. Here, we report the atomic- resolution structure of CACTD-SP1 assemblies with the small-molecule maturation inhibitor PF- 46396 and the assembly cofactor inositol hexakisphosphate (IP6), determined by magic angle spinning (MAS) NMR spectroscopy. Our results reveal that although the two PF-46396 enantiomers exhibit distinct binding modes, they both possess similar anti-HIV potency. PF-46396 binding arrests IP6 dynamics in the six-helix bundle pore, and the two enantiomers induce unique IP6 orientations in the pore. Importantly, our data suggest the presence of monoanionic IP6 form IP6 in the complex. Our study establishes the structural basis for PF-46396 action and suggests a mechanistic model for drug resistance.

biophysics↗

bioRxiv2022

Structural Basis of HIV-1 Maturation Inhibitor Binding and Activity

HIV-1 maturation inhibitors (MIs) interfere with the final step in the viral lifecycle by disrupting the ordered proteolytic processing of the viral Gag polyprotein into its individual domains. Bevirimat (BVM) and its analogs interfere with the final catalytic cleavage of spacer peptide 1 (SP1) from the capsid protein (CA) C-terminal domain (CACTD), by binding to and stabilizing the CACTD-SP1 region. MIs are under development as alternative drugs to augment current antiretroviral therapies. Although promising, their mechanism of action and associated virus resistance pathways remain poorly understood at the molecular, biochemical, and structural levels. Here, we report atomic-resolution magic angle spinning (MAS) NMR structures of microcrystalline assemblies of CACTD-SP1 complexed with BVM and/or the assembly cofactor inositol hexakisphosphate (IP6). BVM and IP6 can bind simultaneously to SP1, with BVM positioned in the center of its 6-helix bundle in a unique conformation. Importantly, the NMR-observed structural effects of BVM on IP6 binding suggest that the inhibitor stabilizes the 6-helix bundle in multiple ways. In addition, BVM-resistant SP1-A1V and SP1-V7A variants exhibit distinct conformational and binding characteristics. Taken together, our results reveal a novel allosteric mechanism by which BVM disrupts maturation and provide a structural explanation for BVM resistance as well as important guidance for the design of new MIs.

biophysics↗

bioRxiv2020

Permeability of the HIV-1 capsid to metabolites modulates viral DNA synthesis

Reverse transcription, an essential event in the HIV-1 lifecycle, requires deoxynucleotide triphosphates (dNTPs) to fuel DNA synthesis, thus requiring penetration of dNTPs into the viral core. The central cavity of the capsid protein (CA) hexamer reveals itself as a plausible channel that allows the passage of dNTPs into assembled capsids. Nevertheless, the molecular mechanism of nucleotide import into the capsid remains unknown. Employing all-atom molecular dynamics simulations, we established that cooperative binding between nucleotides inside a CA hexamer cavity results in energetically-favorable conditions for passive translocation of dNTPs into the HIV-1 capsid. Furthermore, binding of the host cell metabolite inositol hexakisphosphate (IP6) enhances dNTP import, while binding of synthesized molecules like benzenehexacarboxylic acid (BHC) inhibits it. The enhancing effect on reverse transcription by IP6 and the consequences of interactions between CA and nucleotides were corroborated using atomic force microscopy, transmission electron microscopy, and virological assays. Collectively, our results provide an atomistic description of the permeability of the HIV-1 capsid to small molecules and reveal a novel mechanism for the involvement of metabolites in HIV-1 capsid stabilization, nucleotide import and reverse transcription.

biophysics↗

bioRxiv2019

Conformational Flexibility of p150Glued(1-191) Subunit of Dynactin Assembled with Microtubules

Microtubule-associated proteins (MAPs) perform diverse functions in cells. These functions are dependent on their interactions with microtubules. Dynactin, a cofactor of dynein motor, assists the binding of dynein to various organelles and is crucial to the long-distance processivity of dynein-based complexes. The largest subunit of dynactin, the p150glued, contains a N-terminus segment that is responsible for the microtubule-binding interactions and long-range processivity of dynactin. We employed solution and magic angle spinning NMR spectroscopy to characterize the structure and dynamics of the p150glued N-terminal region, both free and in complex with polymerized microtubules. This 191-residue region encompasses the CAP-Gly domain, the basic domain and serine-proline-rich (SP-rich) domain. We demonstrate that the basic and SP-rich domains are intrinsically disordered in solution and significantly enhance the binding affinity to microtubules as these regions contain the second microtubule-binding site on the p150Glued subunit. The majority of the basic and SP-rich domains are predicted to be random-coil, while the segments S111-I116, A124-R132 and K144-T146 in the basic domain contain short -helical or {beta}-sheet structures. These three segments possibly encompass the microtubule binding site. Surprisingly, the protein retains high degree of flexibility upon binding to microtubules except for the regions that are directly involved in the binding interactions with microtubules. This conformational flexibility may be essential for the biological functions of the p150Glued subunit.\n\nSTATEMENT OF SIGNIFICANCEMicrotubule-associated proteins (MAPs) perform diverse functions in cells. Many of them comprises intrinsically disordered regions, whose structural flexibility are central to microtubule-based cellular functions of MAPs. We employed solution and magic angle spinning NMR spectroscopy to characterize the structure and dynamics of the p150glued N-terminal region encompassing the CAP-Gly domain, the basic domain and serine-proline-rich (SP-rich) domain, both free and in complex with polymerized microtubules. The results reveal that the basic and SP-rich domains are largely unstructured and retains high degree of flexibility upon binding to microtubules except for the regions that are possibly involved in the binding interactions with microtubules. This approach is informative for dynamics studies of intrinsically disordered MAPs and other disordered proteins in large biological assemblies.

biophysics↗