Swinger Kerren K, Rice Phoebe A
Department of Biochemistry and Molecular Biology, University of Chicago, 920 E 58th Street, Chicago, IL 60637, USA.
J Mol Biol. 2007 Jan 26;365(4):1005-16. doi: 10.1016/j.jmb.2006.10.024. Epub 2006 Oct 13.
HU and IHF are prokaryotic proteins that induce very large bends in DNA. They are present in high concentrations in the bacterial nucleoid and aid in chromosomal compaction. They also function as regulatory cofactors in many processes, such as site-specific recombination and the initiation of replication and transcription. HU and IHF have become paradigms for understanding DNA bending and indirect readout of sequence. While IHF shows significant sequence specificity, HU binds preferentially to certain damaged or distorted DNAs. However, none of the structurally diverse HU substrates previously studied in vitro is identical with the distorted substrates in the recently published Anabaena HU(AHU)-DNA cocrystal structures. Here, we report binding affinities for AHU and the DNA in the cocrystal structures. The binding free energies for formation of these AHU-DNA complexes range from approximately 10-14.5 kcal/mol, representing K(d) values in the nanomolar to low picomolar range, and a maximum stabilization of at least approximately 6.3 kcal/mol relative to complexes with undistorted, non-specific DNA. We investigated IHF binding and found that appropriate structural distortions can greatly enhance its affinity. On the basis of the coupling of structural and relevant binding data, we estimate the amount of conformational strain in an IHF-mediated DNA kink that is relieved by a nick (at least 0.76 kcal/mol) and pinpoint the location of the strain. We show that AHU has a sequence preference for an A+T-rich region in the center of its DNA-binding site, correlating with an unusually narrow minor groove. This is similar to sequence preferences shown by the eukaryotic nucleosome.
HU和IHF是能在DNA中诱导产生很大弯曲的原核蛋白。它们在细菌类核中高浓度存在，有助于染色体压缩。它们在许多过程中还作为调节辅因子发挥作用，如位点特异性重组以及复制和转录的起始。HU和IHF已成为理解DNA弯曲和序列间接识别的范例。虽然IHF表现出显著的序列特异性，但HU优先结合某些受损或扭曲的DNA。然而，之前在体外研究的结构多样的HU底物中，没有一个与最近发表的鱼腥藻HU（AHU）-DNA共晶体结构中的扭曲底物相同。在此，我们报告了AHU与共晶体结构中DNA的结合亲和力。这些AHU-DNA复合物形成的结合自由能范围约为10 - 14.5千卡/摩尔，代表纳摩尔到低皮摩尔范围内的K(d)值，相对于与未扭曲的非特异性DNA形成的复合物，最大稳定化至少约为6.3千卡/摩尔。我们研究了IHF的结合情况，发现适当的结构扭曲可极大增强其亲和力。基于结构和相关结合数据的耦合，我们估计了由一个切口缓解的IHF介导的DNA扭结中的构象应变大小（至少0.76千卡/摩尔），并确定了应变的位置。我们表明AHU在其DNA结合位点中心对富含A+T的区域有序列偏好，这与异常狭窄的小沟相关。这与真核核小体表现出的序列偏好相似。
