Biology subjects

Sivaramakrishnan, S.

Publications and source records attributed to Sivaramakrishnan, S..

5 recordsLinked to original sources

bioRxiv2023

Tail Length and E525K Dilated Cardiomyopathy Mutant Alter Human β-Cardiac Myosin Super-Relaxed State

Dilated cardiomyopathy (DCM) is characterized by impaired cardiac function due to myocardial hypo-contractility and is associated with point mutations in {beta}-cardiac myosin, the molecular motor that powers cardiac contraction. Myocardial function can be modulated through sequestration of myosin motors into an auto-inhibited "super relaxed" state (SRX), which is further stabilized by a structural state known as the "Interacting Heads Motif" (IHM). Therefore, hypo-contractility of DCM myocardium may result from: 1) reduced function of individual myosin, and/or; 2) decreased myosin availability due to increased IHM/SRX stabilization. To define the molecular impact of an established DCM myosin mutation, E525K, we characterized the biochemical and mechanical activity of wild-type (WT) and E525K human {beta}-cardiac myosin constructs that differed in the length of their coiled-coil tail, which dictates their ability to form the IHM/SRX state. Single-headed (S1) and a short-tailed, double-headed (2HEP) myosin constructs exhibited low ([~]10%) IHM/SRX content, actin-activated ATPase activity of [~]5s-1 and fast velocities in unloaded motility assays ([~]2000nm/s). Double-headed, longer-tailed (15HEP, 25HEP) constructs exhibited higher IHM/SRX content ([~]90%), and reduced actomyosin ATPase (<1s-1) and velocity ([~]800nm/s). A simple analytical model suggests that reduced velocities may be attributed to IHM/SRX-dependent sequestration of myosin heads. Interestingly, the E525K 15HEP and 25HEP mutants showed no apparent impact on velocity or actomyosin ATPase at low ionic strength. However, at higher ionic strength, the E525K mutation stabilized the IHM/SRX state. Therefore, the E525K-associated DCM human cardiac hypo-contractility may be attributable to reduced myosin head availability caused by enhanced IHM/SRX stability. SummaryThis research investigates the E525K mutation in human {beta}-cardiac myosin, crucial for heart contraction, and its role in causing Dilated Cardiomyopathy (DCM). It demonstrates that the length of the myosin tail influences its self-inhibition, and the E525K mutation strengthens this effect, potentially reducing heart contractility in DCM.

molecular biology↗

bioRxiv2023

Cellular Lipids Regulate the Conformational Ensembles of the Disordered Intracellular Loop 3 in beta-2 Adrenergic Receptor

The structurally disordered intracellular loops (ICLs) of G protein-coupled receptors (GPCRs) play a critical role in G protein coupling. In our previous work, we used a combination of FRET-based and computational methodologies to show that the third intracellular loop (ICL3) modulates the activity and G protein coupling selectivity in GPCRs. In the current study, we have uncovered the role of several lipid components in modulating the conformational ensemble of ICL3 of the {beta}2-adrenergic receptor ({beta}2AR). Our findings indicate that phosphatidylinositol 4,5-bisphosphate (PIP2) in the inner leaflet of the membrane bilayer acts as a stabilizing anchor for ICL3, opening the intracellular cavity to facilitate G protein coupling. This interaction between PIP2 and ICL3 causes tilting of {beta}2AR within the cellular membrane. Notably, this tilting of the receptor is supported by ganglioside GM3 stabilizing the extracellular loops on the outer leaflet of the bilayer, thereby exerting an allosteric effect on the orthosteric ligand binding pocket. Our results underscore the significance of lipids in modulating GPCR activity, proposing an allosteric mechanism that occurs through the receptors orientation within the membrane.

biophysics↗

bioRxiv2022

Dissecting β-Cardiac Myosin and Cardiac Myosin-Binding Protein C Interactions using a Nanosurf Assay

Cardiac myosin-binding protein C (cMyBP-C) regulates cardiac contractility by slowing shortening velocity and sensitizing the thin filament to calcium. cMyBP-C has been shown to interact with the proximal myosin S2 tail and the thin filament. However, the relative contribution of these interactions to the collective modulation of actomyosin ensemble function remains unclear. Hence, we developed a "nanosurf" assay as a model system to interrogate cMyBP-C interactions with actin and/or myosin. Synthetic thick filaments were generated using recombinant human {beta}-cardiac myosin subfragments (HMM or S1) attached to DNA nanotubes, with 14 or 28 nm spacing, corresponding to the 14.3 nm myosin spacing found in native thick filaments. In vitro motility assays with myosin bound to the surrounding surface, exhibit enhanced thin filament interactions with synthetic thick filaments. No significant differences were observed in mean thin filament velocities between 14 and 28 nm spacing, consistent with our previous results for myosin V, VI, and {beta}-cardiac myosin S1. Our nanosurf assay demonstrates the slowing of actomyosin motility by cMyBP-C. Alternating {beta}-cardiac myosin HMM and cMyBP-C N-terminal fragments, C0-C2 or C1-C2, every 14 nm on the nanotube, reduced the mean thin filament velocity 4-6 fold relative to myosin alone. Interestingly, similar inhibition was observed using a {beta}-cardiac myosin S1 construct, which lacks the S2 region proposed to interact with cMyBP-C, suggesting the actin-cMyBP-C interactions may dominate the inhibitory mechanism. No significant inhibition of thin filament velocity was observed with a C0-C1f fragment, lacking the majority of the M-domain, supporting the importance of this domain for inhibitory interaction(s). A phosphomimetic C0-C2 fragment showed a 3-fold higher velocity compared to its phosphonull counterpart, further highlighting phosphorylation-dependent regulation via the M-domain. Together, we have established the nanosurf assay as a tool to precisely manipulate spatially dependent cMyBP-C binding partner interactions, shedding light on the molecular regulation of {beta}-cardiac myosin contractility. STATEMENT OF SIGNIFICANCECardiac myosin-binding protein C (cMyBP-C) is the most frequently mutated protein associated with hypertrophic cardiomyopathy (HCM), a common cause of sudden cardiac death. Despite the importance of cMyBP-C in cardiac contractility, the mechanisms underlying this regulation are unclear due to experimental challenges in studying the complex, transient, weak interactions of cMyBP-C with the contractile proteins of the sarcomere. In this study, we created a nanosurf synthetic DNA thick filament assay to dissect the cMyBP-C interactions with actin and human {beta}-cardiac myosin. We demonstrate actomyosin inhibition by cMyBP-C fragments regardless of recombinant human {beta}-cardiac myosin subfragment (HMM or S1) and highlight the importance of the cMyBP-C M-domain using cMyBP-C fragments and phosphomimetics.

biophysics↗

bioRxiv2022

Dilated cardiomyopathy mutation E525K in human beta-cardiac myosin stabilizes the interacting heads motif and super-relaxed state of myosin

The auto-inhibited, super-relaxed (SRX) state of cardiac myosin is thought to be crucial for regulating contraction, relaxation, and energy conservation in the heart. We used single ATP turnover experiments to demonstrate that a dilated cardiomyopathy (DCM) mutation (E525K) in human beta-cardiac myosin increases the fraction of myosin heads in the SRX state (with slow ATP turnover), especially in physiological ionic strength conditions. We also utilized FRET between a C-terminal GFP tag on the myosin tail and Cy3ATP bound to the active site of the motor domain to estimate the fraction of heads in the closed, interacting-heads motif (IHM); we found a strong correlation between the IHM and SRX state. Negative stain EM and 2D class averaging of the construct demonstrated that the E525K mutation increased the fraction of molecules adopting the IHM. Overall, our results demonstrate that the E525K DCM mutation may reduce muscle force and power by stabilizing the auto-inhibited SRX state. Our studies also provide direct evidence for a correlation between the SRX biochemical state and the IHM structural state in cardiac muscle myosin. Furthermore, the E525 residue may be implicated in crucial electrostatic interactions that modulate this conserved, auto-inhibited conformation of myosin. Significance StatementDilated cardiomyopathy can be caused by single point mutations in cardiac muscle myosin, the motor protein that powers contraction of the myocardium. We found that the E525K DCM mutation in the cardiac myosin heavy chain stabilizes the auto-inhibited, super-relaxed state, suggesting a mechanism by which this mutation reduces muscle force and power. The E525K mutation also highlights critical electrostatic interactions important for forming the conserved, auto-inhibited conformational state of striated muscle myosins.

biophysics↗

bioRxiv2021

The Effect of Ligands and Transducers on the Neurotensin Recep-tor 1 (NTS1) Conformational Ensemble

Using a discrete, intracellular 19F-NMR probe on transmembrane helix 6 (TM6) of the Neurotensin receptor 1 (NTS1), we aim to understand how ligands and transducers modulate the receptors structural ensemble in solution. For apo NTS1, 19F-NMR spectra reveal an ensemble of at least three conformational substates (one inactive and two active-like) in equilibrium that exchange on the ms-s timescale. Dynamic NMR experiments reveal that these substates follow a linear three-site exchange process that is both thermodynamically and kinetically remodeled by orthosteric ligands. As previously observed in other GPCRs, the full agonist is insufficient to completely stabilize the active-like state. The inactive substate is abolished upon coupling to {beta}-arrestin-1 or the C-terminal helix of Gq, which comprises 60% of the GPCR/G protein interface surface area. Whereas {beta}-arrestin-1 exclusively selects for pre-existing active-like substates, the Gq peptide induces a new substate. Both transducer molecules promote substantial line-broadening of active-like states suggesting contributions from additional s-ms exchange processes. Together, our study suggests i) the NTS1 allosteric activation mechanism may be alternatively dominated by induced fit or conformational selection depending on the coupled transducer, and ii) the available static structures do not represent the entire conformational ensemble observed in solution.

biophysics↗