Biology subjects

Ha, B. H.

Publications and source records attributed to Ha, B. H..

5 recordsLinked to original sources

bioRxiv2026

Cryo-EM structure and biochemical characterization of a BRAF/CRAF heterodimer: Negative charge in the NtA motif is not required for RAF activation

Upon RAS-driven membrane recruitment, RAF kinases ARAF, BRAF and CRAF are activated via formation of homo- or hetero-dimers to initiate signaling through the MAP kinase cascade. Although RAF heterodimers are important for both physiologic and oncogenic signaling, they have been little studied at a structural and biochemical level. Here we report the preparation, biochemical characterization, and the cryo-EM structure of a 14-3-3-bound BRAF/CRAF heterodimer complex. The heterodimer exhibited kinetic parameters and sensitivity to a panel of twelve structurally diverse RAF inhibitors that were closely similar to, or intermediate between, those of BRAF and CRAF homodimers. Cryo-EM structures of the heterodimer with and without MEK1 revealed an overall organization essentially identical to that of RAF homodimers, but with an asymmetric interaction in the MEK1-bound structure in which the BRAF N-terminal acidic (NtA) motif extends across the dimer interface to engage the CRAF RKTR motif. Mutagenesis of this interface unexpectedly revealed that replacing the acidic NtA sequence with a basic RARA sequence yields highly active RAF homodimers and heterodimers, demonstrating that negative charge in the NtA motif is not required for activity. Collectively, our findings suggest that the charge state of the NtA motif influences RAF activity through effects on local backbone dynamics and the stability of the inactive kinase conformation, rather than via stereospecific recognition across the dimer interface.

biophysics↗

bioRxiv2025

RAF isoform selectivity of MEK inhibitors and rational design of a covalent ARAF-MEK inhibitor

Aberrant activation of the RAS/RAF/MEK/ERK pathway is a frequent cause of cancer. Allosteric MEK inhibitors block this pathway by binding RAF-MEK complexes to prevent activation of MEK by RAF. However, how MEK inhibitor potency varies across the three RAF isoforms remains poorly understood. We profiled seven allosteric MEK inhibitors and observed a striking hierarchy of sensitivity: all most potently inhibited CRAF-driven MEK activation while relatively sparing ARAF-driven activation. We identified point mutations in ARAF and CRAF proximate to the MEK inhibitor binding site that markedly altered inhibitor sensitivity. Using a rational design approach, we developed a more potent inhibitor of ARAF-driven MEK signaling, TWG-07-148. Our cryo-EM structure shows how this acrylamide-containing analog of MEK inhibitor trametinib covalently targets Cys514, a residue unique to ARAF. Our studies highlight the importance of the activating RAF isoform as a determinant of MEK inhibitor sensitivity and provide proof-of-concept for development of MEK inhibitors that more effectively block ARAF-driven MEK signaling via covalent targeting of Cys514.

biochemistry↗

bioRxiv2025

In vitro reconstitutions suggest a general model for paradoxical activation of ARAF, BRAF, and CRAF by diverse RAF inhibitor types that does not rely on negative allostery.

RAF kinases are central regulators of the RAS/MAP kinase pathway and important targets in cancer therapy. Paradoxically, RAF inhibitors can activate wild-type RAF signaling. Negative allostery is a central feature of the prevailing model for this phenomenon, wherein inhibitors induce RAF dimers in which inhibitor binding to one protomer promotes an active but inhibitor-resistant conformation in the other protomer. Here we systematically examined paradoxical activation of ARAF, BRAF, and CRAF using biochemical assays with isolated RAF/MEK kinase domain complexes. We found that type I and type II inhibitors induce paradoxical activation of all three isoforms, and that phosphomimetic mutation of the N-terminal acidic motif of ARAF and CRAF dramatically sensitized these isoforms to activation by type II inhibitors. The inhibition phase of paradoxical activation curves for type II inhibitors was suggestive of positive cooperativity, a finding in conflict with the prevailing model which implies negative cooperativity. In contrast to the kinase domain RAF/MEK preparations, full-length autoinhibited RAF/MEK/14-3-3 complexes were refractory to activation. Mass photometry confirmed that paradoxical activators promote BRAF dimerization. These findings support a revised model that does not rely on negative allostery. Inhibitors act on the RAS-engaged "open monomer" state to induce dimerization and activation. The open monomer and active dimer are structurally distinct species with differing affinities for inhibitor and ATP, creating a concentration window in which paradoxical activation occurs.

biochemistry↗

bioRxiv2025

Characterization and inhibitor sensitivity of ARAF, BRAF, and CRAF complexes

The RAS-RAF-MEK-ERK signaling pathway controls cellular growth and proliferation, and mutational activation of this pathway is a frequent cause of cancer. Most prominently, the V600E mutation in BRAF causes malignant melanoma, papillary thyroid cancer and other malignancies. Rare but recurrent activating mutations in the other two RAF isoforms, ARAF and CRAF, have also been identified in diverse cancers. Distinct classes of RAF inhibitors have been developed, particularly for BRAFV600E, but their potencies against the three RAF isoforms have not been systematically compared. Here we biochemically characterize monomeric and dimeric preparations of ARAF, BRAF, and CRAF and measure the potencies of a panel of thirteen type I, type I.5, and type II RAF inhibitors against each active RAF preparation. Type I inhibitor SB590885 is roughly equipotent across RAF isoforms and, as expected, type I.5 inhibitors are typically most potent against BRAFV600E. Despite their reputation as pan-RAF inhibitors, type II inhibitors as a class are potent inhibitors of CRAF but exhibit relative sparing of ARAF and intermediate potencies against BRAF. Type II compounds inhibit BRAF and CRAF with marked positive cooperativity, and their apparent potencies are insensitive to ATP-concentrations. Crystal structures of CRAF in complex with type I.5 inhibitor PLX4720 reveal an asymmetric CRAF dimer with one CRAF subunit bound in the inactive state and the second bound in an C-helix-in, active conformation with an altered inhibitor pose. Our findings have important implications for understanding the pharmacology of current RAF inhibitors and will inform development of new agents with distinct isoform selectivity.

biochemistry↗

bioRxiv2025

Nickel binding to a split ATCUN motif in c-Src SH3 domain facilitates crystallization

Numerous X-ray crystal structures of the c-Src SH3 domain have provided a large sampling of atomic-level information for this important signaling domain. Multiple crystal forms have been reported, with variable crystal lattice contacts and chemical crystallization conditions. Here, we report a unique crystal structure of Src SH3 domain in trigonal space group H32 to 1.45 [A] resolution. Crystal packing and anomalous scattering reveal that this crystal form is mediated by two ordered nickel ions provided by the crystallization buffer. Nickel coordination occurs in a 2:2 stoichiometry which dimerizes two SH3 domain monomers across a pseudo-twofold rotation axis and involves the native N-terminal c-Src SH3 amino acid sequence, a surface-exposed histidine residue, and ordered water molecules. This study provides an example of metal binding by N-terminal protein residues that contrasts the amino terminal copper and nickel binding (ATCUN) motif and is an alternative avenue for crystallization of the Src SH3 domain. STRUCTURED ABSTRACTO_ST_ABSIntroductionC_ST_ABSNumerous X-ray crystal structures of the c-Src SH3 domain have provided a large sampling of atomic-level information for this important signaling domain. Multiple crystal forms have been reported, with variable crystal lattice contacts and chemical crystallization conditions. Materials and MethodsWe crystallize the c-Src SH3 domain in a crystallization buffer containing NiCl2. ResultsA unique crystal structure of Src SH3 domain in trigonal space group H32 to 1.45 [A] resolution is determined. Crystal packing and anomalous scattering reveal that this crystal form is mediated by two ordered nickel ions provided by the crystallization buffer. Nickel coordination occurs in a 2:2 stoichiometry which dimerizes two SH3 domain monomers across a pseudo-twofold rotation axis and involves the native N-terminal c-Src SH3 amino acid sequence, a surface-exposed histidine residue, and ordered water molecules. DiscussionThis study provides an example of metal-mediated crystallization and metal binding by N-terminal protein residues that contrasts the amino terminal copper and nickel binding (ATCUN) motif. ConclusionAlternative avenues for helps widen the potential for future crystallography-based studies of the c-Src SH3 domain.

biochemistry↗