Krishna Chinthalapudi

Mechanism and specificity of pentachloropseudilin-mediated inhibition of myosin motor activity

Here, we report that the natural compound pentachloropseudilin (PClP) acts as a reversible and allosteric inhibitor of myosin ATPase and motor activity. IC50 values are in the range from 1 to 5 μm for mammalian class-1 myosins and greater than 90 μm for class-2 and class-5 myosins, and no inhibition was observed with class-6 and class-7 myosins. We show that in mammalian cells, PClP selectively inhibits myosin-1c function. To elucidate the structural basis for PClP-induced allosteric coupling and isoform-specific differences in the inhibitory potency of the compound, we used a multifaceted approach combining direct functional, crystallographic, and in silico modeling studies. Our results indicate that allosteric inhibition by PClP is mediated by the combined effects of global changes in protein dynamics and direct communication between the catalytic and allosteric sites via a cascade of small conformational changes along a conserved communication pathway.

Lipid binding promotes oligomerization and focal adhesion activity of vinculin

PIP2 binds vinculin and directs its oligomerization, which promotes proper focal adhesion structure and function.

Inhibition of Myosin ATPase activity by halogenated pseudilins: a structure-activity study.

Myosin activity is crucial for many biological functions. Strong links have been established between changes in the activity of specific myosin isoforms and diseases such as cancer, cardiovascular failure, and disorders of sensory organs and the central nervous system. The modulation of specific myosin isoforms therefore holds a strong therapeutic potential. In recent work, we identified members of the marine alkaloid family of pseudilins as potent inhibitors of myosin-dependent processes. Here, we report the crystal structure of the complex between the Dictyostelium myosin 2 motor domain and 2,4-dichloro-6-(3,4,5-tribromo-1H-pyrrole-2-yl)phenol (3). Detailed comparison with previously solved structures of the myosin 2 complex with bound pentabromopseudilin (2a) or pentachloropseudilin (4a) provides insights into the molecular basis of the allosteric communication between the catalytic and the allosteric sites. Moreover, we describe the inhibitory potency for a congeneric series of halogenated pseudilins. Insight into their mode of action is gained by applying a combination of experimental and computational approaches.

Unusual anchor of a motor complex (MyoD-MLC2) to the plasma membrane of Toxoplasma gondii.

Toxoplasma gondii possesses 11 rather atypical myosin heavy chains. The only myosin light chain described to date is MLC1, associated with myosin A, and contributing to gliding motility. In this study, we examined the repertoire of calmodulin‐like proteins in Apicomplexans, identified six putative myosin light chains and determined their subcellular localization in T. gondii and Plasmodium falciparum. MLC2, only found in coccidians, is associated with myosin D via its calmodulin (CaM)‐like domain and anchored to the plasma membrane of T. gondii via its N‐terminal extension. Molecular modeling suggests that the MyoD–MLC2 complex is more compact than the reported structure of Plasmodium MyoA–myosin A tail‐interacting protein (MTIP) complex. Anchorage of this MLC2 to the plasma membrane is likely governed by palmitoylation.

Kinetic characterization of the sole nonmuscle myosin-2 from the model organism Drosophila melanogaster

Nonmuscle myosin‐2 is the primary enzyme complex powering contractility of the F‐actin cytoskeleton in the model organism Drosophila. Despite myosins essential function in fly development and homeostasis, its kinetic features remain elusive. The purpose of this in vitro study is a detailed steady‐state and presteady‐state kinetic characterization of the Drosophila nonmuscle myosin‐2 motor domain. Kinetic features are a slow steady‐state ATPase activity, high affinities for F‐actin and ADP, and a low duty ratio. Comparative analysis of the overall enzymatic signatures across the nonmuscle myosin‐2 complement from model organisms indicates that the Drosophila protein resembles nonmuscle myosin‐2s from metazoa rather than protozoa, though modulatory aspects of myosin motor function are distinct. Drosophila nonmuscle myosin‐2 is uniquely insensitive toward blebbistatin, a commonly used myosin‐2 inhibitor. An in silico modeling approach together with kinetic studies indicate that the nonconsensus amino acid Met466 in the Drosophila nonmuscle myosin‐2 active‐site loop switch‐2 acts as blebbistatin desensitizer. Introduction of the M466I mutation sensitized the protein for blebbistatin, resulting in a half‐maximal inhibitory concentration of 36.3 ± 4.1 μM. Together, these data show that Drosophila nonmuscle myosin‐2 is a bona fide molecular motor and establish an important link between switch‐2 and blebbistatin sensitivity.—Heissler, S. M., Chinthalapudi, K., Sellers, J. R. Kinetic characterization of the sole nonmuscle myosin‐2 from the model organism Drosophila melanogaster. FASEB J. 29, 1456‐1466 (2015). www.fasebj.org

Lipid-directed vinculin dimerization.

Vinculin localizes to cellular adhesions where it regulates motility, migration, development, wound healing, and response to force. Importantly, vinculin loss results in cancer phenotypes, cardiovascular disease, and embryonic lethality. At the plasma cell membrane, the most abundant phosphoinositide, phosphatidylinositol 4,5-bisphosphate (PIP2), binds the vinculin tail domain, Vt, and triggers homotypic and heterotypic interactions that amplify binding of vinculin to the actin network. Binding of PIP2 to Vt is necessary for maintaining optimal focal adhesions, for organizing stress fibers, for cell migration and spreading, and for the control of vinculin dynamics and turnover of focal adhesions. While the recently determined Vt/PIP2 crystal structure revealed the conformational changes occurring upon lipid binding and oligomerization, characterization of PIP2-induced vinculin oligomerization has been challenging in the adhesion biology field. Here, via a series of novel biochemical assays not performed in previous studies that relied on chemical cross-linking, we characterize the PIP2-induced vinculin oligomerization. Our results show that Vt/PIP2 forms a tight dimer with Vt or with the muscle-specific vinculin isoform, metavinculin, at sites of adhesion at the cell membrane. Insight into how PIP2 regulates clustering and into mechanisms that regulate cell adhesion allows the development for a more definite sensor for PIP2, and our developed techniques can be applied generally and thus open the door for the characterization of many other protein/PIP2 complexes under physiological conditions.

Mechanistic insights into the active site and allosteric communication pathways in human nonmuscle myosin-2C

Despite a generic, highly conserved motor domain, ATP turnover kinetics and their activation by F-actin vary greatly between myosin-2 isoforms. Here, we present a 2.25 Å pre-powerstroke state (ADP⋅VO4) crystal structure of the human nonmuscle myosin-2C motor domain, one of the slowest myosins characterized. In combination with integrated mutagenesis, ensemble-solution kinetics, and molecular dynamics simulation approaches, the structure reveals an allosteric communication pathway that connects the distal end of the motor domain with the active site. Disruption of this pathway by mutation of hub residue R788, which forms the center of a cluster of interactions connecting the converter, the SH1-SH2 helix, the relay helix, and the lever, abolishes nonmuscle myosin-2 specific kinetic signatures. Our results provide insights into structural changes in the myosin motor domain that are triggered upon F-actin binding and contribute critically to the mechanochemical behavior of stress fibers, actin arcs, and cortical actin-based structures.

Differential lipid binding of vinculin isoforms promotes quasi-equivalent dimerization.

Significance Debilitating heart conditions, dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM), are often due to inherited or acquired mutations in genes that encode specific components of adhesion complexes. In muscle tissue, some of these adhesion complexes have specialized structures, called intercalated discs, which are important for contraction and coordinated movement. Here we provide molecular insights into the cytoskeletal protein metavinculin, which is necessary for the proper development and maintenance of heart tissue and is mutated in human DCM and HCM. We show that the binding of lipid causes metavinculin to dimerize and involves a specific metavinculin amino acid associated with severe DCM/HCM. Collectively, our studies provide insight into how such metavinculin mutations in components of adhesion complexes lead to cardiomyopathies. The main cause of death globally remains debilitating heart conditions, such as dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM), which are often due to mutations of specific components of adhesion complexes. Vinculin regulates these complexes and plays essential roles in intercalated discs that are necessary for muscle cell function and coordinated movement and in the development and function of the heart. Humans bearing familial or sporadic mutations in vinculin suffer from chronic, progressively debilitating DCM that ultimately leads to cardiac failure and death, whereas autosomal dominant mutations in vinculin can also provoke HCM, causing acute cardiac failure. The DCM/HCM-associated mutants of vinculin occur in the 68-residue insert unique to the muscle-specific, alternatively spliced isoform of vinculin, termed metavinculin (MV). Contrary to studies that suggested that phosphoinositol-4,5-bisphosphate (PIP2) only induces vinculin homodimers, which are asymmetric, we show that phospholipid binding results in a domain-swapped symmetric MV dimer via a quasi-equivalent interface compared with vinculin involving R975. Although one of the two PIP2 binding sites is preserved, the symmetric MV dimer that bridges two PIP2 molecules differs from the asymmetric vinculin dimer that bridges only one PIP2. Unlike vinculin, wild-type MV and the DCM/HCM-associated R975W mutant bind PIP2 in their inactive conformations, and R975W MV fails to dimerize. Mutating selective vinculin residues to their corresponding MV residues, or vice versa, switches the isoform’s dimeric constellation and lipid binding site. Collectively, our data suggest that MV homodimerization modulates microfilament attachment at muscular adhesion sites and furthers our understanding of MV-mediated cardiac remodeling.

Lipid binding promotes the open conformation and tumor-suppressive activity of neurofibromin 2

Neurofibromatosis type 2 (NF2) is a tumor-forming disease of the nervous system caused by deletion or by loss-of-function mutations in NF2, encoding the tumor suppressing protein neurofibromin 2 (also known as schwannomin or merlin). Neurofibromin 2 is a member of the ezrin, radixin, moesin (ERM) family of proteins regulating the cytoskeleton and cell signaling. The correlation of the tumor-suppressive function and conformation (open or closed) of neurofibromin 2 has been subject to much speculation, often based on extrapolation from other ERM proteins, and controversy. Here we show that lipid binding results in the open conformation of neurofibromin 2 and that lipid binding is necessary for inhibiting cell proliferation. Collectively, our results provide a mechanism in which the open conformation is unambiguously correlated with lipid binding and localization to the membrane, which are critical for the tumor-suppressive function of neurofibromin 2, thus finally reconciling the long-standing conformation and function debate.Neurofibromin 2 (NF2) is a tumour suppressor that inhibits cell growth. Here the authors combine functional, biochemical, and structural studies and show that lipid-bound NF2 adopts an open conformation and that NF2 lipid binding is required for inhibition of cell proliferation.

Kinetic signatures of myosin-5B, the motor involved in microvillus inclusion disease

Myosin-5B is a ubiquitous molecular motor that transports cargo vesicles of the endomembrane system in intracellular recycling pathways. Myosin-5B malfunction causes the congenital enteropathy microvillus inclusion disease, underlining its importance in cellular homeostasis. Here we describe the interaction of myosin-5B with F-actin, nucleotides, and the pyrazolopyrimidine compound myoVin-1. We show that single-headed myosin-5B is an intermediate duty ratio motor with a kinetic ATPase cycle that is rate-limited by the release of phosphate. The presence of a second head generates strain and gating in the myosin-5B dimer that alters the kinetic signature by reducing the actin-activated ADP release rate to become rate-limiting. This kinetic transition into a high-duty ratio motor is a prerequisite for the proposed transport function of myosin-5B in cellular recycling pathways. Moreover, we show that the small molecule compound myoVin-1 inhibits the enzymatic and functional activity of myosin-5B in vitro. Partial inhibition of the actin-activated steady-state ATPase activity and sliding velocity suggests that caution should be used when probing the effect of myoVin-1 on myosin-5–dependent transport processes in cells.