Protein–DNA Recognition Triggered by a DNA Conformational Switch

Abstract

How DNA-binding proteins find their target sites remains a fascinating question. Early work on the Lac repressor showed that proteins specifically bind faster than simple diffusion allows. This led to the idea that recognition could be accelerated by combining diffusion with sliding along DNA and hopping between neighboring strands. This, in turn, implied that proteins could interact with DNA in a distinct nonspecific manner. Experimental work has confirmed that proteins can indeed slide along DNA, although typical sliding distances vary from protein to protein. Recent work also indicates that sliding follows the helical grooves of DNA. Crystallography of proteins bound to noncognate sites, NMR spectroscopy, and molecular simulations have all provided data on nonspecific binding, notably suggesting that proteins maintain similar orientations with respect to DNA in nonspecifically and specifically bound states (see, e.g., Refs. [7–9]). However, little is known about the transition between these states, although theoretical studies have suggested that a switching mechanism may exist, possibly involving a protein conformational change. To analyze this problem at the atomic level, we carried out a molecular dynamics (MD) study on the dissociation of a specific protein–DNA complex in water, starting from the bound conformation. We studied the sex-determining region Y (SRY) protein, which affects the gender selection in mammals and is linked to a number of gender-related pathologies. The SRY protein binds in the minor groove of DNA, optimally at an (A/T)AACAAT sequence, and opens the minor groove by partial intercalation of an isoleucine residue (Ile13) between two adjacent AT base pairs and induces local unwinding and bending of DNA away from the protein. Using a specially designed restraint for the minimal atomic distance between any pair of nonhydrogen atoms across the protein–DNA interface (dMIN), we were able to control the dissociation of the SRY protein from a 14-base-pair (bp) DNA oligomer (5’-CCTGCACAAACACC-3’) without biasing the conformational pathway. Using this approach, roughly 0.6 ms of umbrella sampling led to a free-energy profile for the dissociation/association process. This profile showed a free-energy gain of 11.5 kcal mol 1 because of the SRY-DNA binding; this binding process includes passage of an energy barrier of roughly 4 kcalmol 1 at a separation of 4.2–3.5 and a secondary barrier of 2 kcalmol 1 at 3.1 (see Figure S1 in the Supporting Information). We investigated the conformational aspects of this pathway to understand the recognition mechanism. An initial analysis showed that the conformation of the SRY protein remained remarkably stable during its separation from the DNA. Although the Nand C-terminal tails were very flexible, the three-a-helix protein core varied by a root-mean-square deviation (RMSD) of 2.2 at most (see Figure S1 in the Supporting Information). In contrast, the DNA oligomer underwent considerable change, linked to the SRY-induced deformations. However, our initial analysis of the separation profile also showed that many DNA conformations and protein locations occurred for a single minimal pair distance along the separation pathway. We reduced this problem by using the distance dAXC from the center of the DNA helical axis to the center of mass of the core region of the SRY protein. This distance varies almost monotonically with the minimal pair distance for dMIN 20 . The most interesting feature lies between these two regions (13 > dAXC> 20 ), where the DNA conformations clearly split into two paths (green and red). The center of the two-path region occurs around dAXC= 16 . Here, both paths are found at a RMSD value of around 3 relative to the bound DNA reference state, whereas the upper path (path 1) is located at a RMSD value of 5.2 relative to the unbound [*] Dr. B. Bouvier, Dr. K. Zakrzewska, Dr. R. Lavery Universit Lyon 1/CNRS, UMR 5086, Bases Mol culaires et Structurales des Syst mes Infectieux, IBCP 7 passage du Vercors, 69367 Lyon (France) E-mail: richard.lavery@ibcp.fr Homepage: http://www.ibcp.fr