Enhancing ferryl accumulation in H2O2-dependent cytochrome P450s.

A facile strategy is presented to enhance the accumulation of ferryl (iron(IV)-oxo) species in H2O2 dependent cytochrome P450s (CYPs) of the CYP152 family. We report the characterization of a highly chemoselective CYP decarboxylase from Staphylococcus aureus (OleTSA) that is soluble at high concentrations. Examination of OleTSA Compound I (CpdI) accumulation with a variety of fatty acid substrates reveals a dependence on resting spin-state equilibrium. Alteration of this equilibrium through targeted mutagenesis of the proximal pocket favors the high-spin form, and as a result, enhances Cpd-I accumulation to nearly stoichiometric yields.

Conformational Dynamics Allows Sampling of an "Active-like" State by Oncogenic K-Ras-GDP.

Mutations in K-Ras GTPase replacing Gly12 with either Asp or Val are common in cancer. These mutations decelerate intrinsic and catalyzed GTP hydrolysis, leading to accumulation of K-Ras-GTP in cells. Signaling cascades initiated by K-Ras-GTP promote cell proliferation, survival, and invasion. Despite functional differences between the most frequent G12D mutation and the most aggressive and chemotherapy resistant G12V mutation, their long-suspected distinct structural features remain elusive. Using NMR, X-ray structures, and computational methods, we found that oncogenic mutants of K-Ras4B, the predominant splice variant of K-Ras, exhibit distinct conformational dynamics when GDP-bound, visiting the "active-like" conformational state similar to the one observed in GTP-bound K-Ras. This behavior distinguishes G12V from wild type and G12D K-Ras4B-GDP. The likely reason is interactions between the aliphatic sidechain of V12 and the Switch II region of K-Ras4BG12V-GDP, which are distinct in K-Ras4BG12D-GDP. In the X-ray structures, crystal contacts reduce the dynamics of the sidechain at position 12 by stabilizing the Switch I region of the protein. This explains why structural differences between G12V and G12D K-Ras have yet not been reported. Together, our results suggest a previously unknown mechanism of K-Ras activation. This mechanism relies on conformational dynamics caused by specific oncogenic mutations in the GDP-bound state. Our findings also imply that the therapeutic strategies decreasing the level of K-Ras-GTP by interfering with nucleotide exchange or by expediting GTP hydrolysis may work differently in different oncogenic mutants.

Engineered Allosteric Regulation of Protein Function.

Allosteric regulation of proteins has been utilized to study various aspects of cell signaling, from unicellular events to organism-wide phenotypes. However, traditional methods of allosteric regulation, such as constitutively active mutants and inhibitors, lack tight spatiotemporal control. This often leads to unintended signaling consequences that interfere with data interpretation. To overcome these obstacles, researchers employed protein engineering approaches that enable tight control of protein function through allosteric mechanisms. These methods provide high specificity as well as spatial and temporal precision in regulation of protein activity in vitro and in vivo. In this review, we focus on the recent advancements in engineered allosteric regulation and discuss the various bioengineered allosteric techniques available now, from chimeric GPCRs to chemogenetic and optogenetic switches. We highlight the benefits and pitfalls of each of these techniques as well as areas in which future improvements can be made. Additionally, we provide a brief discussion on implementation of engineered allosteric regulation approaches, demonstrating that these tools can shed light on elusive biological events and have the potential to be utilized in precision medicine.

The dynamic nature of the K-Ras/calmodulin complex can be altered by oncogenic mutations.

Oncogenic mutant K-Ras promotes cancer cell proliferation, migration, invasion, and survival by assembling signaling complexes. To date, the functional and structural roles of K-Ras mutations within these complexes are incompletely understood despite their mechanistic and therapeutic significance. Here, we review recent advances in understanding specific binding between K-Ras and the calcium sensor calmodulin. This interaction positively and negatively regulates diverse functions of K-Ras in cancer, suggesting flexibility in K-Ras/calmodulin complex formation. Also, structural data suggest that oncogenic K-Ras likely samples several conformational states, influencing its distinct assemblies with calmodulin and with other proteins. Understanding how K-Ras interacts with calmodulin and with other partners is essential to discovering novel inhibitors of K-Ras in cancer.

The Hypervariable Region of K-Ras4B Governs Molecular Recognition and Function.

The flexible C-terminal hypervariable region distinguishes K-Ras4B, an important proto-oncogenic GTPase, from other Ras GTPases. This unique lysine-rich portion of the protein harbors sites for post-translational modification, including cysteine prenylation, carboxymethylation, phosphorylation, and likely many others. The functions of the hypervariable region are diverse, ranging from anchoring K-Ras4B at the plasma membrane to sampling potentially auto-inhibitory binding sites in its GTPase domain and participating in isoform-specific protein-protein interactions and signaling. Despite much research, there are still many questions about the hypervariable region of K-Ras4B. For example, mechanistic details of its interaction with plasma membrane lipids and with the GTPase domain require further clarification. The roles of the hypervariable region in K-Ras4B-specific protein-protein interactions and signaling are incompletely defined. It is also unclear why post-translational modifications frequently found in protein polylysine domains, such as acetylation, glycation, and carbamoylation, have not been observed in K-Ras4B. Expanding knowledge of the hypervariable region will likely drive the development of novel highly-efficient and selective inhibitors of K-Ras4B that are urgently needed by cancer patients.

A Distal Loop Controls Product Release and Chemo- and Regioselectivity in Cytochrome P450 Decarboxylases.

Cytochrome P450 OleT utilizes hydrogen peroxide (H2O2) to catalyze the decarboxylation or hydroxylation of fatty acid (FA) substrates. Both reactions are initiated through the abstraction of a substrate hydrogen atom by the high-valent iron-oxo intermediate known as Compound I. Here, we specifically probe the influence of substrate coordination on OleT reaction partitioning through the combined use of fluorescent and electron paramagnetic resonance (EPR)-active FA probes and mutagenesis of a structurally disordered F-G loop that is distal from the heme-iron active site. Both probes are efficiently metabolized by OleT and efficiently trigger the formation of Compound I. Transient fluorescence and EPR reveal a slow product release step, mediated by the F-G loop, that limits OleT turnover. A single-amino acid change or excision of the loop reveals that this region establishes critical interactions to anchor FA substrates in place. The stabilization afforded by the F-G loop is essential for regulating regiospecific C-H abstraction and allowing for efficient decarboxylation to occur. These results highlight a regulatory strategy whereby the fate of activated oxygen species can be controlled at distances far removed from the site of chemistry.