Post-translational modification patterns on β-myosin heavy chain are altered in ischemic and non-ischemic human hearts

Phosphorylation and acetylation of sarcomeric proteins are important for fine-tuning myocardial contractility. Here, we used bottom-up proteomics and label-free quantification to identify novel post-translational modifications (PTMs) on beta-myosin heavy chain (β-MHC) in normal and failing human heart tissues. We report six acetylated lysines and two phosphorylated residues: K34-Ac, K58-Ac, S210-P, K213-Ac, T215-P, K429-Ac, K951-Ac, and K1195-Ac. K951-Ac was significantly reduced in both ischemic and non-ischemic failing hearts compared to non-diseased hearts. Molecular dynamics simulations show that K951-Ac may impact stability of thick filament tail interactions and ultimately myosin head positioning. K58-Ac altered the solvent exposed SH3 domain surface – known for protein-protein interactions – but did not appreciably change motor domain conformation or dynamics under conditions studied. Together, K213-Ac/T215-P altered loop 1’s structure and dynamics – known to regulate ADP-release, ATPase activity, and sliding velocity. Our study suggests that β-MHC acetylation levels may be influenced more by the PTM location than the type of heart disease since less protected acetylation sites are reduced in both heart failure groups. Additionally, these PTMs have potential to modulate interactions between β-MHC and other regulatory sarcomeric proteins, ADP-release rate of myosin, flexibility of the S2 region, and cardiac myofilament contractility in normal and heart failure hearts.

Deafness mutation in the MYO3A motor domain impairs actin protrusion elongation mechanism

Class III myosins are actin-based motors proposed to transport cargo to the distal tips of stereocilia in the inner ear hairs cells and/or to participate in stereocilia length regulation, which is especially important during development. Mutations in the MYO3A gene are associated with delayed onset deafness. A previous study demonstrated that L697W, a dominant deafness mutation, disrupts MYO3A ATPase and motor properties but does not impair its ability to localize to the tips of actin protrusions. In the current study, we characterized the transient kinetic mechanism of the L697W motor ATPase cycle. Our kinetic analysis demonstrates that the mutation slows the ADP release and ATP hydrolysis steps, which results in a slight reduction in the duty ratio and slows detachment kinetics. Fluorescence recovery after photobleaching (FRAP) of filopodia tip localized L697W and WT MYO3A in COS-7 cells revealed that the mutant does not alter turnover or average intensity at the actin protrusion tips. We demonstrate that the mutation slows filopodia extension velocity in COS-7 cells which correlates with its 2-fold slower in vitro actin gliding velocity. Overall, this work allowed us to propose a model for how the motor properties of MYO3A are crucial for facilitating actin protrusion length regulation.

Electrostatic interaction of loop 1 and backbone of human cardiac myosin regulates the rate of ATP induced actomyosin dissociation

Double mutation D208Q:K450L was introduced in the beta isoform of human cardiac myosin to remove the salt bridge D208:K450 connecting loop 1 and the seven stranded beta sheet within the myosin head. Beta isoform specific salt bridge D208:K450 was previously discovered in the molecular dynamics simulations. It was proposed that loop 1 modulates nucleotide affinity to actomyosin and we hypothesized that the electrostatic interactions between loop 1 and myosin head backbone regulates ATP binding to and ADP dissociation from actomyosin, and therefore, the time of the strong actomyosin binding. Wild type and the mutant of the myosin head construct (843 amino acid residues) were expressed in differentiated C2C12 cells, and the kinetics of ATP induced actomyosin dissociation and ADP release were characterized using transient kinetics spectrophotometry. Both constructs exhibit a fast rate of ATP binding to actomyosin and a slow rate of ADP dissociation, showing that ADP release limits the time of the strongly bound state of actomyosin. We observed a faster rate of ATP induced actomyosin dissociation with the mutant, compared to the wild type actomyosin. The rate of ADP release from actomyosin remains the same for the mutant and the wild type actomyosin. We conclude that the flexibility of loop 1 is a factor affecting the rate of ATP binding to actomyosin and actomyosin dissociation. We observed no effect of loop 1 flexibility on the rate of ADP release from actomyosin.

Preproteins couple the intrinsic dynamics of SecA to its ATPase cycle to translocate via a catch and release mechanism

Protein machines undergo conformational motions to interact with and manipulate polymeric substrates. The Sec translocase promiscuously recognizes, becomes activated and secretes >500 non-folded preprotein clients across bacterial cytoplasmic membranes. Here, we reveal that the intrinsic dynamics of the translocase ATPase, SecA, and of preproteins combine to achieve translocation. SecA possesses an intrinsically dynamic preprotein clamp attached to an equally dynamic ATPase motor. Alternating motor conformations are finely controlled by the γ-phosphate of ATP, while ADP causes motor stalling, independently of clamp motions. Functional preproteins physically bridge these independent dynamics. Their signal peptide promotes clamp closing; their mature domain overcomes the rate limiting ADP release. While repeated ATP cycles shift the motor between unique states, multiple conformationally frustrated prongs in the clamp repeatedly catch and release trapped preprotein segments until translocation completion. This universal mechanism allows any preprotein to promiscuously recognize the translocase, usurp its intrinsic dynamics and become secreted.

Elucidation of Master Allostery Essential for Circadian Clock Oscillation in Cyanobacteria

Spatio-temporal allostery is the source of complex but ordered biological phenomena. To identify the structural basis for allostery that drives the cyanobacterial circadian clock, we crystallized the clock protein KaiC in four distinct states, which cover a whole cycle of phosphor–transfer events at Ser431 and Thr432. The minimal set of allosteric events required for oscillatory nature is a bidirectional coupling between the coil-to-helix transition of the Ser431-dependent phospho-switch in the C-terminal domain of KaiC and ADP release from its N-terminal domain during ATPase cycle. An engineered KaiC–protein oscillator consisting of a minimal set of the identified master allosteric events exhibited mono-phosphorylation cycle of Ser431 with a temperature-compensated circadian period, providing design principles for simple post-translational biochemical circadian oscillators.One Sentence SummaryCoupling between a phospho-switch and KaiC ATPase-dependent nucleotide exchange drives the cyanobacterial circadian clock.

Identification of sequence changes in myosin II that adjust muscle contraction velocity

The speed of muscle contraction is related to body size; muscles in larger species contract at slower rates. Since contraction speed is a property of the myosin isoform expressed in a muscle, we investigated how sequence changes in a range of muscle myosin II isoforms enable this slower rate of muscle contraction. We considered 798 sequences from 13 mammalian myosin II isoforms to identify any adaptation to increasing body mass. We identified a correlation between body mass and sequence divergence for the motor domain of the 4 major adult myosin II isoforms (β/Type I, IIa, IIb, and IIx), suggesting that these isoforms have adapted to increasing body mass. In contrast, the non-muscle and developmental isoforms show no correlation of sequence divergence with body mass. Analysis of the motor domain sequence of β-myosin (predominant myosin in Type I/slow and cardiac muscle) from 67 mammals from 2 distinct clades identifies 16 sites, out of 800, associated with body mass (padj < 0.05) but not with the clade (padj > 0.05). Both clades change the same small set of amino acids, in the same order from small to large mammals, suggesting a limited number of ways in which contraction velocity can be successfully manipulated. To test this relationship, the 9 sites that differ between human and rat were mutated in the human β-myosin to match the rat sequence. Biochemical analysis revealed that the rat–human β-myosin chimera functioned like the native rat myosin with a 2-fold increase in both motility and in the rate of ADP release from the actin–myosin crossbridge (the step that limits contraction velocity). Thus, these sequence changes indicate adaptation of β-myosin as species mass increased to enable a reduced contraction velocity and heart rate.

Cryo-EM structures reveal how ATP and DNA binding in MutS coordinate the sequential steps of DNA mismatch repair

DNA mismatch repair detects and removes mismatches from DNA reducing the error rate of DNA replication a 100-1000 fold. The MutS protein is one of the key players that scans for mismatches and coordinates the repair cascade. During this, MutS undergoes multiple conformational changes that initiate the subsequent steps, in response to ATP binding, hydrolysis, and release. How ATP induces the different conformations in MutS is not well understood. Here we present four cryo-EM structures of Escherichia coli MutS at sequential stages of the ATP hydrolysis cycle. These structures reveal how ATP binding and hydrolysis induces a closing and opening of the MutS dimer, respectively. Additional biophysical analysis furthermore explains how DNA binding modulates the ATPase cycle by preventing hydrolysis during scanning and mismatch binding, while preventing ADP release in the sliding clamp state. Nucleotide release is achieved when MutS encounters single stranded DNA that is produced during the removal of the daughter strand. This way, the combination of the ATP binding and hydrolysis and its modulation by DNA enable MutS to adopt different conformations needed to coordinate the sequential steps of the mismatch repair cascade.

The ATPase mechanism of myosin 15, the molecular motor mutated in DFNB3 human deafness

Cochlear hair cells each possess an exquisite bundle of actin-based stereocilia that detect sound. Unconventional myosin 15 (MYO15A) traffics and delivers critical molecules required for stereocilia development and thus is essential for building the mechanosensory hair bundle. Mutations in the human MYO15A gene interfere with stereocilia trafficking and cause hereditary hearing loss, DFNB3, but the impact of these mutations is not known, as MYO15A itself is poorly characterized. To learn more, we performed a kinetic study of the ATPase motor domain to characterize its mechano-chemical cycle. Using the baculovirus-Sf9 system, we purified a recombinant minimal motor domain (S1) by co-expressing the mouse MYO15 ATPase, essential and regulatory light chains that bind its IQ domains, and UNC45 and HSP90A chaperones required for correct folding of the ATPase. MYO15 purified with either UNC45A or UNC45B co-expression had similar ATPase activities (kcat = ~ 6 s-1 at 20°C). Using stopped-flow and quenched-flow transient kinetic analyses, we measured the major rate constants describing the ATPase cycle, including ATP , ADP and actin binding, hydrolysis and phosphate release. Actin-attached ADP release was the slowest measured transition (~ 12 s-1 at 20°C), although this did not rate-limit the ATPase cycle. The kinetic analysis shows the MYO15 motor domain has a moderate duty ratio (~ 0.5) and weak thermodynamic coupling between ADP and actin binding. These findings are consistent with MYO15 being kinetically adapted for processive motility when oligomerized. Our kinetic characterization enables future studies into how deafness-causing mutations affect MYO15 and disrupt stereocilia trafficking necessary for hearing.

Unraveling a Force-Generating Allosteric Pathway of Actomyosin Communication Associated with ADP and Pi Release

The actomyosin system generates mechanical work with the execution of the power stroke, an ATP-driven, two-step rotational swing of the myosin-neck that occurs post ATP hydrolysis during the transition from weakly to strongly actin-bound myosin states concomitant with Pi release and prior to ADP dissociation. The activating role of actin on product release and force generation is well documented; however, the communication paths associated with weak-to-strong transitions are poorly characterized. With the aid of mutant analyses based on kinetic investigations and simulations, we identified the W-helix as an important hub coupling the structural changes of switch elements during ATP hydrolysis to temporally controlled interactions with actin that are passed to the central transducer and converter. Disturbing the W-helix/transducer pathway increased actin-activated ATP turnover and reduced motor performance as a consequence of prolonged duration of the strongly actin-attached states. Actin-triggered Pi release was accelerated, while ADP release considerably decelerated, both limiting maximum ATPase, thus transforming myosin-2 into a high-duty-ratio motor. This kinetic signature of the mutant allowed us to define the fractional occupancies of intermediate states during the ATPase cycle providing evidence that myosin populates a cleft-closure state of strong actin interaction during the weak-to-strong transition with bound hydrolysis products before accomplishing the power stroke.

Structural and Computational Insights into a Blebbistatin-Bound Myosin•ADP Complex with Characteristics of an ADP-Release Conformation along the Two-Step Myosin Power Stoke

The motor protein myosin drives a wide range of cellular and muscular functions by generating directed movement and force, fueled through adenosine triphosphate (ATP) hydrolysis. Release of the hydrolysis product adenosine diphosphate (ADP) is a fundamental and regulatory process during force production. However, details about the molecular mechanism accompanying ADP release are scarce due to the lack of representative structures. Here we solved a novel blebbistatin-bound myosin conformation with critical structural elements in positions between the myosin pre-power stroke and rigor states. ADP in this structure is repositioned towards the surface by the phosphate-sensing P-loop, and stabilized in a partially unbound conformation via a salt-bridge between Arg131 and Glu187. A 5 Å rotation separates the mechanical converter in this conformation from the rigor position. The crystallized myosin structure thus resembles a conformation towards the end of the two-step power stroke, associated with ADP release. Computationally reconstructing ADP release from myosin by means of molecular dynamics simulations further supported the existence of an equivalent conformation along the power stroke that shows the same major characteristics in the myosin motor domain as the resolved blebbistatin-bound myosin-II·ADP crystal structure, and identified a communication hub centered on Arg232 that mediates chemomechanical energy transduction.