Hypertrophic cardiomyopathy mutations Y115H and E497D disrupt the folded-back state of human beta-cardiac myosin allosterically.

At the molecular level, clinical hypercontractility associated with many hypertrophic cardiomyopathy (HCM)-causing mutations in beta-cardiac myosin appears to be driven by their disruptive effect on the energy-conserving, folded-back, super relaxed (SRX) OFF-state of myosin. A pathological increase in force production results from release of heads from this OFF-state, which results in an increase in the number of heads free to interact with actin and produce force. Pathogenic mutations in myosin can conceivably disrupt the OFF-state by (1) directly affecting the intramolecular interfaces stabilizing the folded-back state, or (2) allosterically destabilizing the folded-back state via disruption of diverse conformational states of the myosin motor along its chemomechanical cycle. However, very little is understood about the mutations that fall in the latter group. Here, using recombinant human beta-cardiac myosin, we analysed the biomechanical properties of two such HCM-causing mutations, Y115H (in the transducer) and E497D (in the relay helix), neither of which falls in the regions that interact to stabilize the myosin folded-back state. We find these mutations have diverse effects on the contractility parameters of myosin, yet the primary hypercontractile change in both cases is the destabilization of the OFF-state of myosin and increased availability of active myosin heads for actin-binding. Experimental data and molecular dynamics simulations indicate that these mutations likely destabilize the pre-powerstroke state of myosin, the conformation the motor adopts in the inactive folded-back state. We propose that destabilization of the folded-back state of myosin, directly and/or allosterically, is the molecular basis of hypercontractility in HCM in a far greater number of pathogenic mutations than currently thought.

Cryo-EM structure of the folded-back state of human ß-cardiac myosin.

To save energy and precisely regulate cardiac contractility, cardiac muscle myosin heads are sequestered in an 'off' state that can be converted to an 'on' state when exertion is increased. The 'off' state is equated with a folded-back structure known as the interacting-heads motif (IHM), which is a regulatory feature of all class-2 muscle and non-muscle myosins. We report here the human ß-cardiac myosin IHM structure determined by cryo-electron microscopy to 3.6 Å resolution, providing details of all the interfaces stabilizing the 'off' state. The structure shows that these interfaces are hot spots of hypertrophic cardiomyopathy mutations that are thought to cause hypercontractility by destabilizing the 'off' state. Importantly, the cardiac and smooth muscle myosin IHM structures dramatically differ, providing structural evidence for the divergent physiological regulation of these muscle types. The cardiac IHM structure will facilitate development of clinically useful new molecules that modulate IHM stability.

Cryo-EM structure of the folded-back state of human ß-cardiac myosin.

During normal levels of exertion, many cardiac muscle myosin heads are sequestered in an off-state even during systolic contraction to save energy and for precise regulation. They can be converted to an on-state when exertion is increased. Hypercontractility caused by hypertrophic cardiomyopathy (HCM) myosin mutations is often the result of shifting the equilibrium toward more heads in the on-state. The off-state is equated with a folded-back structure known as the interacting head motif (IHM), which is a regulatory feature of all muscle myosins and class-2 non-muscle myosins. We report here the human ß-cardiac myosin IHM structure to 3.6 Å resolution. The structure shows that the interfaces are hot spots of HCM mutations and reveals details of the significant interactions. Importantly, the structures of cardiac and smooth muscle myosin IHMs are dramatically different. This challenges the concept that the IHM structure is conserved in all muscle types and opens new perspectives in the understanding of muscle physiology. The cardiac IHM structure has been the missing puzzle piece to fully understand the development of inherited cardiomyopathies. This work will pave the way for the development of new molecules able to stabilize or destabilize the IHM in a personalized medicine approach. *This manuscript was submitted to Nature Communications in August 2022 and dealt efficiently by the editors. All reviewers received this version of the manuscript before 9 208 August 2022. They also received coordinates and maps of our high resolution structure on the 18 208 August 2022. Due to slowness of at least one reviewer, this contribution was delayed for acceptance by Nature Communications and we are now depositing in bioRxiv the originally submitted version written in July 2022 for everyone to see. Indeed, two bioRxiv contributions at lower resolution but adding similar concepts on thick filament regulation were deposited this week in bioRxiv, one of the contributions having had access to our coordinates. We hope that our data at high resolution will be helpful for all readers that appreciate that high resolution information is required to build accurate atomic models and discuss implications for sarcomere regulation and the effects of cardiomyopathy mutations on heart muscle function.

Nanomechanical Phenotypes in Cardiac Myosin-Binding Protein C Mutants That Cause Hypertrophic Cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is a disease of the myocardium caused by mutations in sarcomeric proteins with mechanical roles, such as the molecular motor myosin. Around half of the HCM-causing genetic variants target contraction modulator cardiac myosin-binding protein C (cMyBP-C), although the underlying pathogenic mechanisms remain unclear since many of these mutations cause no alterations in protein structure and stability. As an alternative pathomechanism, here we have examined whether pathogenic mutations perturb the nanomechanics of cMyBP-C, which would compromise its modulatory mechanical tethers across sliding actomyosin filaments. Using single-molecule atomic force spectroscopy, we have quantified mechanical folding and unfolding transitions in cMyBP-C domains targeted by HCM mutations that do not induce RNA splicing alterations or protein thermodynamic destabilization. Our results show that domains containing mutation R495W are mechanically weaker than wild-type at forces below 40 pN and that R502Q mutant domains fold faster than wild-type. None of these alterations are found in control, nonpathogenic variants, suggesting that nanomechanical phenotypes induced by pathogenic cMyBP-C mutations contribute to HCM development. We propose that mutation-induced nanomechanical alterations may be common in mechanical proteins involved in human pathologies.

A five-residue motif for the design of domain swapping in proteins.

Domain swapping is the process by which identical monomeric proteins exchange structural elements to generate dimers/oligomers. Although engineered domain swapping is a compelling strategy for protein assembly, its application has been limited due to the lack of simple and reliable design approaches. Here, we demonstrate that the hydrophobic five-residue 'cystatin motif' (QVVAG) from the domain-swapping protein Stefin B, when engineered into a solvent-exposed, tight surface loop between two ß-strands prevents the loop from folding back upon itself, and drives domain swapping in non-domain-swapping proteins. High-resolution structural studies demonstrate that engineering the QVVAG stretch independently into various surface loops of four structurally distinct non-domain-swapping proteins enabled the design of different modes of domain swapping in these proteins, including single, double and open-ended domain swapping. These results suggest that the introduction of the QVVAG motif can be used as a mutational approach for engineering domain swapping in diverse ß-hairpin proteins.

Amino-acid composition after loop deletion drives domain swapping.

Rational engineering of a protein to enable domain swapping requires an understanding of the sequence, structural and energetic factors that favor the domain-swapped oligomer over the monomer. While it is known that the deletion of loops between ß-strands can promote domain swapping, the spliced sequence at the position of the loop deletion is thought to have a minimal role to play in such domain swapping. Here, two loop-deletion mutants of the non-domain-swapping protein monellin, frame-shifted by a single residue, were designed. Although the spliced sequence in the two mutants differed by only one residue at the site of the deletion, only one of them (YEIKG) promoted domain swapping. The mutant containing the spliced sequence YENKG was entirely monomeric. This new understanding that the domain swapping propensity after loop deletion may depend critically on the chemical composition of the shortened loop will facilitate the rational design of domain swapping.

Rapidly fatal myeloproliferative disorders in mice with deletion of Casitas B-cell lymphoma (Cbl) and Cbl-b in hematopoietic stem cells.

Casitas B-cell lymphoma (Cbl)-family E3 ubiquitin ligases are negative regulators of tyrosine kinase signaling. Recent work has revealed a critical role of Cbl in the maintenance of hematopoietic stem cell (HSC) homeostasis, and mutations in CBL have been identified in myeloid malignancies. Here we show that, in contrast to Cbl or Cbl-b single-deficient mice, concurrent loss of Cbl and Cbl-b in the HSC compartment leads to an early-onset lethal myeloproliferative disease in mice. Cbl, Cbl-b double-deficient bone marrow cells are hypersensitive to cytokines, and show altered biochemical response to thrombopoietin. Thus, Cbl and Cbl-b play redundant but essential roles in HSC regulation, whose breakdown leads to hematological abnormalities that phenocopy crucial aspects of mutant Cbl-driven human myeloid malignancies.

Reciprocal regulation of AKT and MAP kinase dictates virus-host cell fusion.

Viruses of the Paramyxoviridae family bind to their host cells by using hemagglutinin-neuraminidase (HN), which enhances fusion protein (F)-mediated membrane fusion. Although respiratory syncytial virus and parainfluenza virus 5 of this family are suggested to trigger host cell signaling during infection, the virus-induced intracellular signals dictating virus-cell fusion await elucidation. Using an F- or HN-F-containing reconstituted envelope of Sendai virus, another paramyxovirus, we revealed the role and regulation of AKT1 and Raf/MEK/ERK cascades during viral fusion with liver cells. Our observation that extracellular signal-regulated kinase (ERK) activation promotes viral fusion via ezrin-mediated cytoskeletal rearrangements, whereas AKT1 attenuates fusion by promoting phosphorylation of F protein, indicates a counteractive regulation of viral fusion by reciprocal activation of AKT1 and mitogen-activated protein kinase (MAPK) cascades, establishing a novel conceptual framework for a therapeutic strategy.