{"doi":"10.1002/cac2.12395","title":"Activation mechanisms of clinically distinct B‐Raf V600E and V600K mutants","abstract":"Dear Editor, B-Raf, the main effector of Ras in the mitogen-activated protein kinase (MAPK) pathway, is among the most highly mutated kinases in human cancer [1]. About 40%-60% of melanoma patients harbor B-Raf mutations, of which ∼90% involve V600E and V600K. B-RafV600E is more frequent (60%-80%) than B-RafV600K (10%-30%). Substitution of a Val codon by Glu requires a single nucleotide change, whereas Val to Lys requires two [2]. This is in line with melanoma patients harboring the V600K mutation, who usually suffer from higher sun exposure that may induce increased DNA damage [3]. Since both mutations occur at the same position of the kinase domain and are mutated to charged residues, it was believed that the B-Raf V600E and V600K mutants would share a similar behavior, and in clinical trials, patients with V600E and V600K mutations have been recruited into the same cohort. However, emerging data suggest that B-Raf V600E and V600K mutants are not identical [4]. V600K tumors are more aggressive than V600E. Patients harboring V600K have a higher risk for relapse and shorter survival than those with V600E [5]. V600K tumors are less responsive to some kinase inhibitors but benefit from immunotherapy treatments [6]. These results imply that the two mutants may have intrinsic molecular differences. However, little is known about the underlying mechanism and the structural basis mediating these differences. B-Raf V600E and V600K mutants can be constitutively activated as monomers [7]. On the other hand, it has been shown that the dimerization of B-RafV600 mutants does take place and is important for MAPK signal transduction in cancer [8]. Thus, our premise was that while B-RafV600 mutants can be active as monomers, their dimerization can enhance their signaling. To explore both scenarios, we studied B-Raf V600E and V600K mutants in the monomeric, homo-dimeric, and hetero-dimeric states at atomic resolution, aiming to identify structural features that may differentiate between B-Raf V600E and V600K mutants and help clarify their clinical outcomes. The study methods are described in the Supplementary Materials. The V600 residue plays essential roles in maintaining the inactive conformation of B-Raf's kinase domain [9, 10]. Its mutations to E600 or K600 result in two structural changes involving (i) longer side-chain and (ii) loss of hydrophobicity. Our modeling shows that the long side-chains in the B-Raf V600E and V600K mutants caused structural collapse of the N-lobe hydrophobic surface in the inactive OFF-state conformation. The charged side-chains of E600 and K600 in the mutants disfavor hydrophobic residues in this region (Supplementary Figure S1). These structural effects may synergistically destabilize the inactive OFF-state conformation, shifting the populations towards the active state. In contrast, in the active ON-state conformation, the mutations to E600 and K600 led to neither structural collapse nor disfavored residue contacts. The E600 and K600 in the B-Raf mutants approached the bottom region of the αC-helix, pointing away from the ATP pocket. This can be confirmed by the crystal structures of the B-RafV600E mutants in the Protein Data Bank (PDB). Except for structures with large inhibitors that prevent the “IN” αC-helix, the B-RafV600E mutants adopt the active ON-state conformation (Supplementary Figure S2). These observations indicate that both B-Raf V600E and V600K mutants favor the active ON-state conformation. In the active ON-state conformation, the monomeric B-Raf V600E and V600K mutants exhibited notable structural differences in local residue contacts (Figure 1A). In B-RafV600E, the negatively charged E600 residue formed a salt bridge with the positively charged K507 residue in the αC-helix's basic box, 506RKTR509, resembling the pS602 in the active wild-type B-Raf [10] (Figure 1B). It also frequently interacted with another basic residue, R603, in the activation loop (A-loop), which may help stabilize the ne","journal":"Cancer Communications","year":2022,"id":263833,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":13,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9552,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2022-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":643963,"name":"Ryan Maloney","orcid":"0000-0001-5876-9371","position":1,"is_corresponding":false},{"id":856358,"name":"Yonglan Liu","orcid":"0000-0001-5280-5992","position":2,"is_corresponding":false},{"id":249368,"name":"Hyunbum Jang","orcid":"0000-0001-9402-4051","position":3,"is_corresponding":false},{"id":70301,"name":"Ruth Nussinov","orcid":"0000-0002-8115-6415","position":4,"is_corresponding":false},{"id":249367,"name":"Mingzhen Zhang","orcid":"0000-0003-3437-2884","position":0,"is_corresponding":true}],"reference_count":10,"raw_metadata":null,"created_at":"2026-07-19T00:26:33.413850Z","pmid":"36573259","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}