Disulfide engineering of the FANCM – MHF complex reveals constraints on crosslink design in symmetric oligomers

Abstract

The Fanconi anemia complementation group M protein (FANCM)–MHF complex is required for branched DNA recognition in the Fanconi anemia pathway, but structural analysis of the intact complex has been hindered by dissociation of FANCM from the FANCM‐associated histone fold (MHF) heterotetramer under crystallization conditions. Here, we used structure‐guided disulfide engineering to stabilize the FANCM–MHF interface and test whether local geometry is sufficient to predict crosslinking specificity in a symmetric oligomeric assembly. Using endogenous FANCM Cys759 as an anchor, we designed two MHF2 variants, Q74C and L77C. Both supported oxidation‐dependent crosslinking in the context of the FANCM–MHF complex, but with distinct outcomes. Q74C formed the intended FANCM–MHF2 disulfide, enabled crystallization of the intact heteropentamer, and preserved DNA‐binding behavior under the tested conditions. In contrast, L77C favored a competing MHF2‐MHF2 disulfide and yielded only the MHF heterotetramer after FANCM dissociation. Structural analysis further showed distinct crosslinking states for the two MHF tetramers in the asymmetric unit, consistent with local conformational heterogeneity at the MHF dimer–dimer interface. These results show that geometric plausibility alone does not predict crosslinking specificity in symmetric oligomers. Instead, symmetry‐related competing pathways can redirect the reaction toward an alternative assembly state. This study provides a practical route to stabilizing FANCM–MHF and reveals a key design constraint for engineered disulfides in symmetric multimeric assemblies.