Solution structure of the Mu end DNA‐binding Iβ subdomain of phage Mu transposase: modular DNA recognition by two tethered domains

The phage Mu transposase (MuA) binds to the ends of the Mu genome during the assembly of higher order nucleoprotein complexes. We investigate the structure and function of the MuA end‐binding domain (Iβγ). The three‐dimensional solution structure of the Iβ subdomain (residues 77–174) has been determined using multidimensional NMR spectroscopy. It comprises five α‐helices, including a helix–turn–helix (HTH) DNA‐binding motif formed by helices 3 and 4, and can be subdivided into two interacting structural elements. The structure has an elongated disc‐like appearance from which protrudes the recognition helix of the HTH motif. The topology of helices 2–4 is very similar to that of helices 1–3 of the previously determined solution structure of the MuA Iγ subdomain and to that of the homeodomain family of HTH DNA‐binding proteins. We show that each of the two subdomains binds to one half of the 22 bp recognition sequence, Iβ to the more conserved Mu end distal half (β subsite) and Iγ to the Mu end proximal half (γ subsite) of the consensus Mu end‐binding site. The complete Iβγ domain binds the recognition sequence with a 100‐ to 1000‐fold higher affinity than the two subdomains independently, indicating a cooperative effect. Our results show that the Mu end DNA‐binding domain of MuA has a modular organization, with each module acting on a specific part of the 22 bp binding site. Based on the present binding data and the structures of the Iβ and Iγ subdomains, a model for the interaction of the complete Iβγ domain with DNA is proposed.