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.

Omicron Binding Mode: Contact Analysis and Dynamics of the Omicron Receptor-Binding Domain in Complex with ACE2.

On 26 November 2021, the WHO classified the Omicron variant of the SARS-CoV-2 virus (B.1.1.529 lineage) as a variant of concern (VOC) (COVID-19 Variant Data, Department of Health, 2022). The Omicron variant contains as many as 26 unique mutations of effects not yet determined (Venkatakrishnan, A., Open Science Framework, 2021). Out of its total of 34 Spike protein mutations, 15 are located on the receptor-binding domain (S-RBD) (Stanford Coronavirus Antiviral & Resistance Database, 2022) that directly contacts the angiotensin-converting enzyme 2 (ACE2) host receptor and is also a primary target for antibodies. Here, we studied the binding mode of the S-RBD domain of the Spike protein carrying the Omicron mutations and the globular domain of human ACE2 using molecular dynamics (MD) simulations. We identified new and key Omicron-specific interactions such as R493 (of mutation Q493R), which forms salt bridges both with E35 and D38 of ACE2, Y501 (N501Y), which forms an edge-to-face aromatic interaction with Y41, and Y505 (Y505H), which makes an H-bond with E37 and K353. The glycan chains of ACE2 also bind differently in the WT and Omicron variants in response to different charge distributions on the surface of Spike proteins. However, while the Omicron mutations considerably improve the overall electrostatic fit of the two interfaces, the total number of specific and favorable interactions between the two does not increase. The dynamics of the complexes are highly affected too, making the Omicron S-RBD:ACE2 complex more rigid; the two main interaction sites, Patches I and II, isolated in the WT complex, become connected in the Omicron complex through the alternating interaction of R493 and R498 with E35 and D38.

Distinct spatial relationship of the interleukin-9 receptor with interleukin-2 receptor and major histocompatibility complex glycoproteins in human T lymphoma cells.

The interleukin-9 receptor (IL-9R) consists of an α subunit and a γ(c) chain that are shared with other cytokine receptors, including interleukin-2 receptor (IL-2R), an important regulator of T cells. We previously showed that IL-2R is expressed in common clusters with major histocompatibility complex (MHC) glycoproteins in lipid rafts of human T lymphoma cells, which raised the question about what the relationship between clusters of IL-2R/MHC and IL-9R is. Confocal microscopy colocalization and fluorescence resonance energy transfer experiments capable of detecting membrane protein organization at different size scales revealed nonrandom association of IL-9R with IL-2R/MHC clusters at the surface of human T lymphoma cells. Accommodation of IL-9Rα in membrane areas segregated from the IL-2R/MHC domains was also detected. The bipartite nature of IL-9R distribution was mirrored by signal transducer and activator of transcription (STAT) activation results. Our data indicate that co-compartmentalization with MHC glycoproteins is a general property of γ(c) receptors. Distribution of receptor chains between different membrane domains may regulate their function.

Two-sided fluorescence resonance energy transfer for assessing molecular interactions of up to three distinct species in confocal microscopy.

The role of the expression patterns of proteins involved in oncogenesis can be understood after characterizing their multimolecular interactions. Conventional FRET methods permit the analysis of interaction between two molecular species at the most, which necessitates the introduction of new approaches for studying multicomponent signaling complexes. Flow cytometric as well as microscopic donor (dbFRET) and acceptor (abFRET) photobleaching FRET measurements were performed to determine the association states of ErbB2, beta1-integrin, and CD44 receptors. Based on consecutively applied abFRET and dbFRET methods (two-sided FRET), the relationship of beta1-integrin-ErbB2 heteroassociation to ErbB2 homoassociation and of beta1-integrin-ErbB2 heteroassociation to ErbB2-CD44 heteroassociation was studied by correlating pixel-by-pixel FRET values of the corresponding abFRET and dbFRET images in contour plots. Anticorrelation was observed between beta1-integrin-ErbB2 heteroassociation and ErbB2 homoassociation on trastuzumab sensitive N87 and SK-BR-3 cells, while modest positive correlation was found between beta1-integrin-ErbB2 and ErbB2-CD44 heteroassociation on trastuzumab resistant MKN-7 cells. The FRET efficiency values of beta1-integrin-ErbB2 heteroassociation were markedly higher at the focal adhesion regions on attached cells than those measured by flow cytometry on detached cells. In conclusion, we implemented an experimental set-up termed two-sided FRET for correlating two pairwise interactions of three arbitrarily chosen molecular species. On the basis of our results, we assume that the homoassociation state of ErbB2 is dynamically modulated by its interaction with beta1-integrins.

Associations of ErbB2, beta1-integrin and lipid rafts on Herceptin (Trastuzumab) resistant and sensitive tumor cell lines.

ErbB2-mediated transmembrane signaling is a key target of novel anticancer agents such as Herceptin. Our comparison of Herceptin resistant (JIMT-1, MKN-7) and sensitive (SKBR-3, N-87) cell lines demonstrates the importance of ErbB2 association patterns involving integrins and lipid rafts. Flow cytometric FRET and confocal microscopic measurements revealed colocalization and molecular proximity between beta1-integrins and ErbB2, as well as their association with lipid rafts. A weak functional interaction between ErbB2 and beta1-integrin and the fact that ErbB2 did not co-patch with beta1-integrins upon crosslinking imply that ErbB2 and beta1-integrin define two distinct molecular association clusters from a functional point of view. Although Herceptin-sensitive cell lines expressed more ErbB2 and fewer beta1-integrin molecules on their surface than their resistant counterparts, this finding probably does not explain the Herceptin resistant phenotype due to the weak interaction between beta1-integrins and ErbB2. Our results imply that the true significance of the expression profile of proteins involved in oncogenesis can only be understood after characterizing their molecular interactions.