The recognition of local DNA conformation by the human papillomavirus type 6 E2 protein

Abstract

The E2 proteins are transcription/replication factors from papillomaviruses. Human papillomaviruses (HPVs) can be broadly divided in two groups; low-risk HPV subtypes cause benign warts while high-risk HPVs give rise to cervical cancer. Although a range of crystal structures of E2 DNA-binding domains (DBD) from both high- and low-risk HPV subtypes have been reported previously, structures of E2 DBD:DNA complexes have only been available for high-risk HPV18 and bovine papillomavirus (BPV1). In the present study we report the unliganded and DNA complex structures of the E2 DBD from the low-risk HPV6. As in the previous E2–DNA structures, complex formation results in considerable bending of the DNA, which is facilitated by sequences with A:T-rich spacers that adopt a pre-bent conformation. The low-risk HPV6 E2–DNA complex differs from the earlier structures in that minimal deformation of the protein accompanies complex formation. Stopped-flow kinetic studies confirm that both high- and low-risk E2 proteins adapt their structures on binding to DNA, although this is achieved more readily for HPV6 E2. It therefore appears that the higher selectivity of the HPV6 E2 protein may arise from its limited molecular adaptability, a property that might distinguish the behaviour of E2 proteins from high- and low-risk HPV subtypes.

Experimental snapshots of a protein-DNA binding landscape

“…Discussion Specific protein-DNA-binding at equilibrium is often a deceptively simple, two-state process in which only the unbound reagents and the final complex are populated (4)(5)(6)(7)(8). However, the few kinetic mechanisms determined to date revealed a richer picture, with parallel routes and transient intermediates (4)(5)(6)(7)(8).…”

“…However, the few kinetic mechanisms determined to date revealed a richer picture, with parallel routes and transient intermediates (4)(5)(6)(7)(8). In the absence of high-resolution kinetic work, structural studies suggested that multistate routes for protein-DNA-binding start with the formation of nonspecific interactions with the DNA backbone (1,(9)(10)(11)(12)(13)(14)(15)(16)(17), and that some dual-role residues may form transient nonnative interactions along the route (13-15, 17).…”

“…It is, thus, generally accepted that these proteins first bind nonspecifically to genomic DNA which speeds up the search for their target sequence through facilitated diffusion (3). The kinetics of DNA sequence recognition (3,4) often involve multistate kinetic routes with populated intermediates (4)(5)(6)(7)(8). The build up of molecular interactions along multistate routes has been deduced indirectly from structural and thermodynamic studies (1)(2)(3)(9)(10)(11)(12)(13)(14)(15)(16)(17), but kinetic characterization of intermediates and transition state ensembles is still scarce.…”

Transition state for protein–DNA recognition

“…Some proteins diffuse mainly in 1D over long distances (2), whereas others favor a 3D pathway (3). Once at the target site, proteins without an enzymatic activity may bind to DNA along a single kinetic route (4)(5)(6)(7)(8)(9) or parallel routes (10,11). Each route may either be two-state (4-6, 10) or include populated kinetic intermediates (7)(8)(9)(10)(11).…”

“…Once at the target site, proteins without an enzymatic activity may bind to DNA along a single kinetic route (4)(5)(6)(7)(8)(9) or parallel routes (10,11). Each route may either be two-state (4-6, 10) or include populated kinetic intermediates (7)(8)(9)(10)(11). In the final consolidated complex, sequence recognition is mediated by specific contacts between protein residues and DNA bases (direct sequence readout) and by the conformational energetics of noncontacted bases (indirect sequence readout) (12)(13)(14)(15)(16).…”