NMR-Chemical-Shift-Driven Protocol Reveals the Cofactor-Bound, Complete Structure of Dynamic Intermediates of the Catalytic Cycle of Oncogenic KRAS G12C Protein and the Significance of the Mg2+ Ion.

In this work, catalytically significant states of the oncogenic G12C variant of KRAS, those of Mg2+-free and Mg2+-bound GDP-loaded forms, have been determined using CS-Rosetta software and NMR-data-driven molecular dynamics simulations. There are several Mg2+-bound G12C KRAS/GDP structures deposited in the Protein Data Bank (PDB), so this system was used as a reference, while the structure of the Mg2+-free but GDP-bound state of the RAS cycle has not been determined previously. Due to the high flexibility of the Switch-I and Switch-II regions, which also happen to be the catalytically most significant segments, only chemical shift information could be collected for the most important regions of both systems. CS-Rosetta was used to derive an "NMR ensemble" based on the measured chemical shifts, which, however, did not contain the nonprotein components of the complex. We developed a torsional restraint set for backbone torsions based on the CS-Rosetta ensembles for MD simulations, overriding the force-field-based parametrization in the presence of the reinserted cofactors. This protocol (csdMD) resulted in complete models for both systems that also retained the structural features and heterogeneity defined by the measured chemical shifts and allowed a detailed comparison of the Mg2+-bound and Mg2+-free states of G12C KRAS/GDP.

Inhibitor Design Strategy for Myostatin: Dynamics and Interaction Networks Define the Affinity and Release Mechanisms of the Inhibited Complexes.

Myostatin, an important negative regulator of muscle mass, is a therapeutic target for muscle atrophic disorders such as muscular dystrophy. Thus, the inhibition of myostatin presents a strategy to treat these disorders. It has long been established that the myostatin prodomain is a strong inhibitor of the mature myostatin, and the minimum peptide of the prodomain-corresponding to the α1-helix of its lasso-region-responsible for the inhibitory efficiency was defined and characterized as well. Here we show that the minimum peptide segment based on the growth differentiation factor 11 (GDF11), which we found to be more helical in its stand-alone solvated stfate than the similar segment of myostatin, is a promising new base scaffold for inhibitor design. The proposed inhibitory peptides in their solvated state and in complex with the mature myostatin were analyzed by in silico molecule modeling supplemented with the electronic circular dichroism spectroscopy measurements. We defined the Gaussian-Mahalanobis mean score to measure the fraction of dihedral angle-pairs close to the desired helical region of the Ramachandran-plot, carried out RING analysis of the peptide-protein interaction networks and characterized the internal motions of the complexes using our rigid-body segmentation protocol. We identified a variant-11m2-that is sufficiently ordered both in solvent and within the inhibitory complex, forms a high number of contacts with the binding-pocket and induces such changes in its internal dynamics that lead to a rigidified, permanently locked conformation that traps this peptide in the binding site. We also showed that the naturally evolved α1-helix has been optimized to simultaneously fulfill two very different roles: to function as a strong binder as well as a good leaving group. It forms an outstanding number of non-covalent interactions with the mature core of myostatin and maintains the most ordered conformation within the complex, while it induces independent movement of the gate-keeper ß-hairpin segment assisting the dissociation and also results in the least-ordered solvated form which provides extra stability for the dissociated state and discourages rebinding.