A Top-Down Proteomic Assay to Evaluate KRAS4B-Compound Engagement.

Development of new targeted inhibitors for oncogenic KRAS mutants may benefit from insight into how a given mutation influences the accessibility of protein residues and how compounds interact with mutant or wild-type KRAS proteins. Targeted proteomic analysis, a key validation step in the KRAS inhibitor development process, typically involves both intact mass- and peptide-based methods to confirm compound localization or quantify binding. However, these methods may not always provide a clear picture of the compound binding affinity for KRAS, how specific the compound is to the target KRAS residue, and how experimental conditions may impact these factors. To address this, we have developed a novel top-down proteomic assay to evaluate in vitro KRAS4B-compound engagement while assessing relative quantitation in parallel. We present two applications to demonstrate the capabilities of our assay: maleimide-biotin labeling of a KRAS4BG12D cysteine mutant panel and treatment of three KRAS4B proteins (WT, G12C, and G13C) with small molecule compounds. Our results show the time- or concentration-dependence of KRAS4B-compound engagement in context of the intact protein molecule while directly mapping the compound binding site.

Comparative analysis of KRAS4a and KRAS4b splice variants reveals distinctive structural and functional properties.

KRAS, the most frequently mutated oncogene in human cancer, produces two isoforms, KRAS4a and KRAS4b, through alternative splicing. These isoforms differ in exon 4, which encodes the final 15 residues of the G-domain and hypervariable regions (HVRs), vital for trafficking and membrane localization. While KRAS4b has been extensively studied, KRAS4a has been largely overlooked. Our multidisciplinary study compared the structural and functional characteristics of KRAS4a and KRAS4b, revealing distinct structural properties and thermal stability. Position 151 influences KRAS4a's thermal stability, while position 153 affects binding to RAF1 CRD protein. Nuclear magnetic resonance analysis identified localized structural differences near sequence variations and provided a solution-state conformational ensemble. Notably, KRAS4a exhibits substantial transcript abundance in bile ducts, liver, and stomach, with transcript levels approaching KRAS4b in the colon and rectum. Functional disparities were observed in full-length KRAS variants, highlighting the impact of HVR variations on interaction with trafficking proteins and downstream effectors like RAF and PI3K within cells.

Reduced dynamic complexity allows structure elucidation of an excited state of KRASG13D.

Localized dynamics of RAS, including regions distal to the nucleotide-binding site, is of high interest for elucidating the mechanisms by which RAS proteins interact with effectors and regulators and for designing inhibitors. Among several oncogenic mutants, methyl relaxation dispersion experiments reveal highly synchronized conformational dynamics in the active (GMPPNP-bound) KRASG13D, which suggests an exchange between two conformational states in solution. Methyl and 31P NMR spectra of active KRASG13D in solution confirm a two-state ensemble interconverting on the millisecond timescale, with a major Pγ atom peak corresponding to the dominant State 1 conformation and a secondary peak indicating an intermediate state different from the known State 2 conformation recognized by RAS effectors. High-resolution crystal structures of active KRASG13D and KRASG13D-RAF1 RBD complex provide snapshots of the State 1 and 2 conformations, respectively. We use residual dipolar couplings to solve and cross-validate the structure of the intermediate state of active KRASG13D, showing a conformation distinct from those of States 1 and 2 outside the known flexible switch regions. The dynamic coupling between the conformational exchange in the effector lobe and the breathing motion in the allosteric lobe is further validated by a secondary mutation in the allosteric lobe, which affects the conformational population equilibrium.

A humanized nanobody phage display library yields potent binders of SARS CoV-2 spike.

Neutralizing antibodies targeting the SARS-CoV-2 spike protein have shown a great preventative/therapeutic potential. Here, we report a rapid and efficient strategy for the development and design of SARS-CoV-2 neutralizing humanized nanobody constructs with sub-nanomolar affinities and nanomolar potencies. CryoEM-based structural analysis of the nanobodies in complex with spike revealed two distinct binding modes. The most potent nanobody, RBD-1-2G(NCATS-BL8125), tolerates the N501Y RBD mutation and remains capable of neutralizing the B.1.1.7 (Alpha) variant. Molecular dynamics simulations provide a structural basis for understanding the neutralization process of nanobodies exclusively focused on the spike-ACE2 interface with and without the N501Y mutation on RBD. A primary human airway air-lung interface (ALI) ex vivo model showed that RBD-1-2G-Fc antibody treatment was effective at reducing viral burden following WA1 and B.1.1.7 SARS-CoV-2 infections. Therefore, this presented strategy will serve as a tool to mitigate the threat of emerging SARS-CoV-2 variants.

A humanized nanobody phage display library yields potent binders of SARS CoV-2 spike.

Neutralizing antibodies targeting the SARS-CoV-2 spike protein have shown a great preventative/therapeutic potential. Here, we report a rapid and efficient strategy for the development and design of SARS-CoV-2 neutralizing humanized nanobody constructs with sub-nanomolar affinities and nanomolar potencies. CryoEM-based structural analysis of the nanobodies in complex with spike revealed two distinct binding modes. The most potent nanobody, RBD-1-2G(NCATS-BL8125), tolerates the N501Y RBD mutation and remains capable of neutralizing the B.1.1.7 (Alpha) variant. Molecular dynamics simulations provide a structural basis for understanding the neutralization process of nanobodies exclusively focused on the spike-ACE2 interface with and without the N501Y mutation on RBD. A primary human airway air-lung interface (ALI) ex vivo model showed that RBD-1-2G-Fc antibody treatment was effective at reducing viral burden following WA1 and B.1.1.7 SARS-CoV-2 infections. Therefore, this presented strategy will serve as a tool to mitigate the threat of emerging SARS-CoV-2 variants.

The role of G proteins in assembly and function of Kir3 inwardly rectifying potassium channels.

Kir3 channels (also known as GIRK channels) are important regulators of electrical excitability in both cardiomyocytes and neurons. Much is known regarding the assembly and function of these channels and the roles that their interacting proteins play in controlling these events. Further, they are one of the best studied effectors of heterotrimeric G proteins in general and Gßγ subunits in particular. However, our understanding of the roles of multiple Gßγ binding sites on Kir3 channels is still rudimentary. We discuss potential roles for Gßγ in channel assembly and trafficking in addition to their known role in cellular signaling.

Intracellular trafficking and assembly of specific Kir3 channel/G protein complexes.

We have previously demonstrated that Kir3.1 channels and Gbeta1gamma2 subunits initially interact in the endoplasmic reticulum (ER). To elucidate the role that anterograde protein trafficking pathways may play in the formation of these complexes, we used dominant negative (DN) mutants of the small G proteins Sar 1 and various compartment-specific Rabs which impede anterograde protein trafficking at different steps. Sar 1 H79G and Rab 1 S25N mutants efficiently blocked the plasma membrane trafficking of the Kir3.1/Kir3.4 complex however they did not block the Gbeta1gamma2/Kir3.1 interaction as measured using bioluminescence resonance energy transfer (BRET). This interaction was also insensitive to the presence of DN Rabs 2, 6, 8, and 11. These results confirm that Gbetagamma/Kir3 complexes form early during channel biosynthesis and trafficking. Using a combination of BRET, protein complementation assays and co-immunoprecipitation, we demonstrate that Gbeta1-4 can interact with Kir3.1 in the absence of Kir3.4. Gbeta5 does not directly interact with the channel but can still be co-immunoprecipitated as part of a larger complex. The interaction between Gbeta and Kir3.1 was selectively fostered by co-expression with different Ggamma subunits. When Ggamma1 or Ggamma11 was co-expressed with eGFP-Gbeta3 or eGFP-Gbeta4, the interaction with the effector was lost. Kir3.2 was capable of interacting with Gbeta1-3 and not Gbeta4 or Gbeta5. These interactions were again fostered by co-expression with Ggamma and were also insensitive to DN Sar 1 or Rab 1. Taken together, our data show that these "precocious" channel/G protein interactions are specific and may have implications beyond their basic function in signalling events.