Ligand-induced conformational changes in a thermophilic ribose-binding protein

<p>Abstract</p> <p>Background</p> <p>Members of the periplasmic binding protein (PBP) superfamily are involved in transport and signaling processes in both prokaryotes and eukaryotes. Biological responses are typically mediated by ligand-induced conformational changes in which the binding event is coupled to a hinge-bending motion that brings together two domains in a closed form. In all PBP-mediated biological processes, downstream partners recognize the closed form of the protein. This motion has also been exploited in protein engineering experiments to construct biosensors that transduce ligand binding to a variety of physical signals. Understanding the mechanistic details of PBP conformational changes, both global (hinge bending, twisting, shear movements) and local (rotamer changes, backbone motion), therefore is not only important for understanding their biological function but also for protein engineering experiments.</p> <p>Results</p> <p>Here we present biochemical characterization and crystal structure determination of the periplasmic ribose-binding protein (RBP) from the hyperthermophile <it>Thermotoga maritima </it>in its ribose-bound and unliganded state. The <it>T. maritima </it>RBP (tmRBP) has 39% sequence identity and is considerably more resistant to thermal denaturation (^<it>app</it><it>T</it>_{<it>m </it>}value is 108°C) than the mesophilic <it>Escherichia coli </it>homolog (ecRBP) (^<it>app</it><it>T</it>_{<it>m </it>}value is 56°C). Polar ligand interactions and ligand-induced global conformational changes are conserved among ecRBP and tmRBP; however local structural rearrangements involving side-chain motions in the ligand-binding site are not conserved.</p> <p>Conclusion</p> <p>Although the large-scale ligand-induced changes are mediated through similar regions, and are produced by similar backbone movements in tmRBP and ecRBP, the small-scale ligand-induced structural rearrangements differentiate the mesophile and thermophile. This suggests there are mechanistic differences in the manner by which these two proteins bind their ligands and are an example of how two structurally similar proteins utilize different mechanisms to form a ligand-bound state.</p

Following an environmental carcinogen N2-dG adduct through replication: elucidating blockage and bypass in a high-fidelity DNA polymerase

We have investigated how a benzo[a]pyrene-derived N2-dG adduct, 10S(+)-trans-anti-[BP]-N2-dG ([BP]G*), is processed in a well-characterized Pol I family model replicative DNA polymerase, Bacillus fragment (BF). Experimental results are presented that reveal relatively facile nucleotide incorporation opposite the lesion, but very inefficient further extension. Computational studies follow the possible bypass of [BP]G* through the pre-insertion, insertion and post-insertion sites as BF alternates between open and closed conformations. With dG* in the normal B-DNA anti conformation, BP seriously disturbs the polymerase structure, positioning itself either deeply in the pre-insertion site or on the crowded evolving minor groove side of the modified template, consistent with a polymerase-blocking conformation. With dG* in the less prevalent syn conformation, BP causes less distortion: it is either out of the pre-insertion site or in the major groove open pocket of the polymerase. Thus, the syn conformation can account for the observed relatively easy incorporation of nucleotides, with mutagenic purines favored, opposite the [BP]G* adduct. However, with the lesion in the BF post-insertion site, more serious distortions caused by the adduct even in the syn conformation explain the very inefficient extension observed experimentally. In vivo, a switch to a potentially error-prone bypass polymerase likely dominates translesion bypass

Crystal structure of a thermostable Bacillus DNA polymerase l large fragment at 2.1 Å resolution

AbstractBackground: The study of DNA polymerases in the Pol l family is central to the understanding of DNA replication and repair. DNA polymerases are used in many molecular biology techniques, including PCR, which require a thermostable polymerase. In order to learn about Pol l function and the basis of thermostability, we undertook structural studies of a new thermostable DNA polymerase.Results: A DNA polymerase large, Klenow-like, fragment from a recently identified thermostable strain of Bacillus stearothermophilus (BF) was cloned, sequenced, overexpressed and characterized. Its crystal structure was determined to 2.1 Å resolution by the method of multiple isomorphous replacement.Conclusions: This structure represents the highest resolution view of a Pol l enzyme obtained to date. Comparison of the three Pol l structures reveals no compelling evidence for many of the specific interactions that have been proposed to induce thermostability, but suggests that thermostability arises from innumerable small changes distributed throughout the protein structure. The polymerase domain is highly conserved in all three proteins. The N-terminal domains are highly divergent in sequence, but retain a common fold. When present, the 3′-5′ proofreading exonuclease activity is associated with this domain. Its absence is associated with changes in catalytic residues that coordinate the divalent ions required for activity and in loops connecting homologous secondary structural elements. In BF, these changes result in a blockage of the DNA-binding cleft