SAXS capabilities of the SIBYLS beamline are demonstrated quite nicely in recent papers published in September and October.
A paper has been published in The October 2 issue of Cell by Scott Williams et al. that sheds light on a previously missing piece of the double-strand break repair complex MRN (aka Mre11-Rad50-Nbs1). The paper entitled “Nbs1 Flexibly Tethers Ctp1 and Mre11-Rad50 to Coordinate DNA Double-Strand Break Processing and Repair” presents a compelling model of the role of Nbs1 (i.e “N” of the MRN) in coordinating double-strand break processing and repair. The paper was made possible in part by crystal structures and SAXS data of Nbs1 that were collected at the SIBYLS beamline.
Abstract:
The Nijmegen breakage syndrome 1 (Nbs1) subunit of the Mre11-Rad50-Nbs1 (MRN) complex protects genome integrity by coordinating double-strand break (DSB) repair and checkpoint signaling through undefined interactions with ATM, MDC1, and Sae2/Ctp1/CtIP. Here, fission yeast and human Nbs1 structures defined by X-ray crystallography and small angle X-ray scattering (SAXS) reveal Nbs1 cardinal features: fused, extended, FHA-BRCT1-BRCT2 domains flexibly linked to C-terminal Mre11- and ATM-binding motifs. Genetic, biochemical, and structural analyses of an Nbs1-Ctp1 complex show Nbs1 recruits phosphorylated Ctp1 to DSBs via binding of the Nbs1 FHA domain to a Ctp1 pThr-Asp motif. Nbs1 structures further identify an extensive FHA-BRCT interface, a divalent MDC1-binding scaffold, an extended conformational switch, and the molecular consequences associated with cancer predisposing Nijmegen breakage syndrome mutations. Tethering of Ctp1 to a flexible Nbs1 arm suggests a mechanism for restricting DNA end processing and homologous recombination activities of Sae2/Ctp1/CtIP to the immediate vicinity of DSBs.
Abstract:Protein biosynthesis on the ribosome requires repeated cycles of ratcheting, which couples rotation of the two ribosomal subunits with respect to each other, and swiveling of the head domain of the small subunit. However, the molecular basis for how the two ribosomal subunits rearrange contacts with each other during ratcheting while remaining stably associated is not known. Here, we describe x-ray crystal structures of the intact Escherichia coli ribosome, either in the apo-form (3.5 angstrom resolution) or with one (4.0 angstrom resolution) or two (4.0 angstrom resolution) anticodon stem-loop tRNA mimics bound, that reveal intermediate states of intersubunit rotation. In the structures, the interface between the small and large ribosomal subunits rearranges in discrete steps along the ratcheting pathway. Positioning of the head domain of the small subunit is controlled by interactions with the large subunit and with the tRNA bound in the peptidyl-tRNA site. The intermediates observed here provide insight into how tRNAs move into the hybrid state of binding that precedes the final steps of mRNA and tRNA translocation.
Cover Illustration from the September 2009 issue of RNA: Crystal structure of the lysine riboswitch bound to lysine (Protein Data Bank code: 3d0u) Image details show RNA: ribbon; lysine: space-filling representation, red; iridium hexamine ions: space-filling representation, blue.
Abstract:
Riboswitches are metabolite-sensitive elements found in mRNAs that control gene expression through a regulatory secondary structural switch. Along with regulation of lysine biosynthetic genes, mutations within the lysine-responsive riboswitch (L-box) play a role in the acquisition of resistance to antimicrobial lysine analogs. To understand the structural basis for lysine binding, we have determined the 2.8 angstroms resolution crystal structure of lysine bound to the Thermotoga maritima asd lysine riboswitch ligand-binding domain. The structure reveals a complex architecture scaffolding a binding pocket completely enveloping lysine. Mutations conferring antimicrobial resistance cluster around this site as well as highly conserved long range interactions, indicating that they disrupt lysine binding or proper folding of the RNA. Comparison of the free and bound forms by x-ray crystallography, small angle x-ray scattering, and chemical probing reveals almost identical structures, indicating that lysine induces only limited and local conformational changes upon binding.
Bernstein NK, Hammel M, Mani RS, Weinfeld M, Pelikan M, Tainer JA, Glover JN. "Mechanism of DNA substrate recognition by the mammalian DNA repair enzyme, Polynucleotide Kinase." Nucleic Acids Res. 2009 Aug 11. [epub].
In this paper, published online today in Nature Methods, the high-throughput SAXS capabilities of the SIBYLS beamline are demonstrated quite nicely.
We present an efficient pipeline enabling high-throughput analysis of protein structure in solution with small angle X-ray scattering (SAXS). Our SAXS pipeline combines automated sample handling of microliter volumes, temperature and anaerobic control, rapid data collection and data analysis, and couples structural analysis with automated archiving. We subjected 50 representative proteins, mostly from Pyrococcus furiosus, to this pipeline and found that 30 were multimeric structures in solution. SAXS analysis allowed us to distinguish aggregated and unfolded proteins, define global structural parameters and oligomeric states for most samples, identify shapes and similar structures for 25 unknown structures, and determine envelopes for 41 proteins. We believe that high-throughput SAXS is an enabling technology that may change the way that structural genomics research is done.
Hura GL, Menon AL, Hammel M, Rambo RP, Poole II FL, Tsutakawa SE, Jenney Jr FE, Classen S, Frankel KA, Hopkins RC, Yang S, Scott JW, Dillard BD, Adams MWW, & Tainer JA, "Robust, high-throughput solution structural analyses by small angle X-ray scattering (SAXS)" Nature Methods. 2009 Aug;6(8):606-12.
