8EZG

Monobody 12D1 bound to KRAS(G12D)

Summary for 8EZG

• Entry DOI: 10.2210/pdb8ezg/pdb
• Related: 8F0M
• Descriptor: GTPase KRas, Monobody 12D1, GUANOSINE-5'-DIPHOSPHATE, ... (6 entities in total)
• Functional Keywords: signaling protein-de novo protein complex, signaling protein/de novo protein
• Biological source: Homo sapiens (human); More
• Total number of polymer chains: 2
• Total formula weight: 29379.77

Authors

Hattori, T.,Glasser, E.,Akkapeddi, P.,Ketavarapu, G.,Teng, K.W.,Koide, A.,Koide, S. (deposition date: 2022-10-31, release date: 2023-07-19, Last modification date: 2023-10-25)

Primary citation

Akkapeddi, P.,Hattori, T.,Khan, I.,Glasser, E.,Koide, A.,Ketavarapu, G.,Whaby, M.,Zuberi, M.,Teng, K.W.,Lefler, J.,Maso, L.,Bang, I.,Ostrowski, M.C.,O'Bryan, J.P.,Koide, S.
Exploring switch II pocket conformation of KRAS(G12D) with mutant-selective monobody inhibitors.
Proc.Natl.Acad.Sci.USA, 120:e2302485120-e2302485120, 2023

PubMed Abstract: The G12D mutation is among the most common KRAS mutations associated with cancer, in particular, pancreatic cancer. Here, we have developed monobodies, small synthetic binding proteins, that are selective to KRAS(G12D) over KRAS(wild type) and other oncogenic KRAS mutations, as well as over the G12D mutation in HRAS and NRAS. Crystallographic studies revealed that, similar to other KRAS mutant-selective inhibitors, the initial monobody bound to the S-II pocket, the groove between switch II and α3 helix, and captured this pocket in the most widely open form reported to date. Unlike other G12D-selective polypeptides reported to date, the monobody used its backbone NH group to directly recognize the side chain of KRAS Asp12, a feature that closely resembles that of a small-molecule inhibitor, MTRX1133. The monobody also directly interacted with H95, a residue not conserved in RAS isoforms. These features rationalize the high selectivity toward the G12D mutant and the KRAS isoform. Structure-guided affinity maturation resulted in monobodies with low nM values. Deep mutational scanning of a monobody generated hundreds of functional and nonfunctional single-point mutants, which identified crucial residues for binding and those that contributed to the selectivity toward the GTP- and GDP-bound states. When expressed in cells as genetically encoded reagents, these monobodies engaged selectively with KRAS(G12D) and inhibited KRAS(G12D)-mediated signaling and tumorigenesis. These results further illustrate the plasticity of the S-II pocket, which may be exploited for the design of next-generation KRAS(G12D)-selective inhibitors.

PubMed: 37399416
DOI: 10.1073/pnas.2302485120

Experimental method

X-RAY DIFFRACTION (2.52 Å)