Mapping the KRAS proteoform landscape in colorectal cancer identifies truncated KRAS4B that decreases MAPK signaling.

The KRAS gene is one of the most frequently mutated oncogenes in human cancer and gives rise to two isoforms, KRAS4A and KRAS4B. KRAS post-translational modifications (PTMs) have the potential to influence downstream signaling. However, the relationship between KRAS PTMs and oncogenic mutations remains unclear, and the extent of isoform-specific modification is unknown. Here, we present the first top-down proteomics study evaluating both KRAS4A and KRAS4B, resulting in 39 completely characterized proteoforms across colorectal cancer cell lines and primary tumor samples. We determined which KRAS PTMs are present, along with their relative abundance, and that proteoforms of KRAS4A versus KRAS4B are differentially modified. Moreover, we identified a subset of KRAS4B proteoforms lacking the C185 residue and associated C-terminal PTMs. By confocal microscopy, we confirmed that this truncated GFP-KRAS4BC185∗ proteoform is unable to associate with the plasma membrane, resulting in a decrease in mitogen-activated protein kinase signaling pathway activation. Collectively, our study provides a reference set of functionally distinct KRAS proteoforms and the colorectal cancer contexts in which they are present.

Crowder-Induced Conformational Ensemble Shift in Escherichia coli Prolyl-tRNA Synthetase.

The effect of molecular crowding on the structure and function of Escherichia coli prolyl-transfer RNA synthetase (Ec ProRS), a member of the aminoacyl-transfer RNA synthetase family, has been investigated using a combined experimental and theoretical method. Ec ProRS is a multidomain enzyme; coupled-domain dynamics are essential for efficient catalysis. To gain insight into the mechanistic detail of the crowding effect, kinetic studies were conducted with varying concentrations and sizes of crowders. In parallel, spectroscopic and quantum chemical studies were employed to probe the "soft interactions" between crowders and protein side chains. Finally, the dynamics of the dimeric protein was examined in the presence of crowders using a long-duration (70 ns) classical molecular dynamic simulations. The results of the simulations revealed a shift in the conformational ensemble, which is consistent with the preferential exclusion of cosolutes. The "soft interactions" model of the crowding effect also explained the alteration in kinetic parameters. In summary, the study found that the effects of molecular crowding on both conformational dynamics and catalytic function are correlated in the multidomain Ec ProRS, an enzyme that is central to protein synthesis in all living cells. This study affirmed that large and small cosolutes have considerable impacts on the structure, dynamics, and function of modular proteins and therefore must be considered for stabilizing protein-based pharmaceuticals and industrial enzymes.

Precise Characterization of KRAS4B Proteoforms by Combining Immunoprecipitation with Top-Down Mass Spectrometry.

The characterization of biologically relevant post-translational modifications (PTMs) on KRAS4B has historically been carried out through methodologies such as immunoblotting with PTM-specific antibodies or peptide-based proteomic methods. While these methods have the potential to identify a given PTM on KRAS4B, they are incapable of characterizing or distinguishing the different molecular forms or proteoforms of KRAS4B from those of related RAS isoforms. We present a method that combines immunoprecipitation of KRAS4B with top-down mass spectrometry (IP-TDMS), thus enabling the precise characterization of intact KRAS4B proteoforms. We provide detailed protocols for the IP, LC-MS/MS, and data analysis comprising a successful IP-TDMS assay in the contexts of cancer cell lines and tissue samples.

Editing Domain Motions Preorganize the Synthetic Active Site of Prolyl-tRNA Synthetase.

Prolyl-tRNA synthetases (ProRSs) catalyze the covalent attachment of proline onto cognate tRNAs, an indispensable step for protein synthesis in all living organisms. ProRSs are modular enzymes and the "prokaryotic-like" ProRSs are distinguished from "eukaryotic-like" ProRSs by the presence of an editing domain (INS) inserted between motifs 2 and 3 of the main catalytic domain. Earlier studies suggested the presence of coupled-domain dynamics could contribute to catalysis; however, the role that the distal, highly mobile INS domain plays in catalysis at the synthetic active site is not completely understood. In the present study, a combination of theoretical and experimental approaches has been used to elucidate the precise role of INS domain dynamics. Quantum mechanical/molecular mechanical simulations were carried out to model catalytic Pro-AMP formation by Enterococcus faecalis ProRS. The energetics of the adenylate formation by the wild-type enzyme was computed and contrasted with variants containing active site mutations, as well as a deletion mutant lacking the INS domain. The combined results revealed that two distinct types of dynamics contribute to the enzyme's catalytic power. One set of motions is intrinsic to the INS domain and leads to conformational preorganization that is essential for catalysis. A second type of motion, stemming from the electrostatic reorganization of active site residues, impacts the height and width of the energy profile and has a critical role in fine tuning the substrate orientation to facilitate reactive collisions. Thus, motions in a distal domain can preorganize the active site of an enzyme to optimize catalysis.