CHE 598 Seminar: “Using NMR-Guided Molecular Dynamics Simulations to Establish a 3-dimensional Model of Protein-Protein Binding Interfaces”

About the event

Presenter: Garry Smith, VSCEB Ph.D. Candidate

Garry Smith is a 3rd year Ph. D student in Chemical Engineering. He began in Fall 2017 after earning a National Institutes of Health (NIH) Protein Biotechnology Traineeship, providing him with two years of funding and the opportunity to rotate in several labs over the first year. After rotations, he chose to work under Dr. Alla Kostyukova where he studies mechanisms of thin filament length regulation by a muscle protein called leiomodin. His work involves utilizing several techniques including nuclear magnetic resonance, molecular dynamics simulations, and circular dichroism spectroscopy to generate 3D structural models of the binding interfaces of leiomodin and its partners, tropomyosin and actin, and to design and test mutations affecting that binding. He has presented his work both at the 2019 GPSA Research Exposition and the 2019 NIH Protein Biotechnology Annual Symposium, earning 3rd place in the Medical and Life Sciences Category at the former.

Using NMR-Guided Molecular Dynamics Simulations to Establish a 3-dimensional Model of Protein-Protein Binding Interfaces

Striated muscle is composed of organized arrays of basic contractile units, sarcomeres, which must be assembled and maintained for proper muscle function. In sarcomeres, thin and thick filaments, composed primarily of actin and myosin, respectively, slide against each other to create muscle contraction. For this system to perform, thin filament (TF) length must be strictly regulated. Improper lengths of actin-thin filaments are associated with lethal myopathies.

Leiomodin (Lmod) and tropomodulin (Tmod), homologous muscle proteins binding to actin and tropomyosin (Tpm), participate in regulating TF length. Lmod is critical in TF assembly and maintenance; however, unlike Tmod, its specific role has remained unclear. In cells, both are found at the slow-growing pointed end of the TF, and, according to an earlier proposed competition mechanism, the two have opposite effects; Tmod halts TF growth while Lmod allows it.

Using nuclear magnetic resonance (NMR) and molecular dynamics simulations (MDS), we generated a novel structural 3D model of the binding interface between the Tpm-binding site (TpmBS1) of cardiac leiomodin and the N-terminus of striated muscle Tpm. Properties of the peptides representing the binding interface and the complex formed by them prevented the structure from being solved outright by NMR. Therefore, obtained NMR data were used to constrain MDS. By NMR-guided MDS, several binding interface topologies were annealed and run for significant time until a stable structure consistent with all NMR data was obtained.

Our data indicate that the Lmod/Tpm complex only forms at the TF pointed end, where the Tpm N-terminus is not blocked by an adjacent Tpm protomer. These observations, together with data from cardiomyocytes, provide evidence supporting the debated mechanism through which Lmod and Tmod regulate TF lengths by competing for the pointed end. Since TpmBS1 is highly homologous across the Tmod family, this study suggests that TpmBS1 is a universal factor contributing to the specificity of Tmod/Lmod binding to actin filament pointed ends.