Beamline BM14 Highlights 2015 beta

Highlight 1: Identification of Arsenic (As) as a source of experimental phasing power

Serendipitous SAD Solution for DMSO-Soaked SOCS2-ElonginC-ElonginB Crystals Using Covalently Incorporated Dimethylarsenic

Abstract

Suppressor of cytokine signalling 2 (SOCS2) is the substrate-binding component of a Cullin-RING E3 ubiquitin ligase (CRL) complex that targets phosphorylated hormone receptors for degradation by the ubiquitin-proteasome system. As a key regulator of the transcriptional response to growth signals, SOCS2 and its protein complex partners are potential targets for small molecule development. We found that crystals of SOCS2 in complex with its adaptor proteins, Elongin C and Elongin B, underwent a change in crystallographic parameters when treated with dimethyl sulfoxide during soaking experiments. To solve the phase problem for the new crystal form we identified the presence of arsenic atoms in the crystals, a result of covalent modification of cysteines by cacodylate, and successfully extracted anomalous signal from these atoms for experimental phasing. The resulting structure provides a means for solving future structures where the crystals must be treated with DMSO for ligand soaking approaches. Additionally, the conformational changes induced in this structure reveal flexibility within SOCS2 that match those postulated by previous molecular dynamics simulations. This conformational flexibility illustrates how SOCS2 can orient its substrates for successful ubiquitination by other elements of the CRL complex.

Citation

Gadd MS, Bulatov E, Ciulli A (2015)
Serendipitous SAD Solution for DMSO-Soaked SOCS2-ElonginC-ElonginB Crystals Using Covalently Incorporated Dimethylarsenic: Insights into Substrate Receptor Conformational Flexibility in Cullin RING Ligases.
PLoS ONE 10(6): e0131218. doi:10.1371/journal.pone.0131218

molecule visualisation
poster
X-ray diffraction Data and Model Statistics

Highlight 2: Experimental phasing power with weakly diffracting crystal (Resolution 3.4Å; exposure time: 40sec/image)

Crystal Structure of Fad35R from Mycobacterium tuberculosis H37Rv in the Apo-State

Abstract

Fad35R from Mycobacterium tuberculosis binds to the promoter site of Fad35 operon and its DNA binding activities are reduced in the presence of tetracycline and palmitoyl-CoA. We resolved the crystal structure of Fad35R using single-wavelength anomalous diffraction method (SAD). Fad35R comprises canonical DNA binding domain (DBD) and ligand binding domain (LBD), but displays several distinct structural features. Two recognition helices of two monomers in the homodimer are separated by ~ 48 Å and two core triangle-shaped ligand binding cavities are well exposed to solvent. Structural comparison with DesT and QacR structures suggests that ligand binding-induced movement of α7, which adopts a straight conformation in the Fad35R, may be crucial to switch the conformational states between repressive and derepressive forms. Two DBDs are packed asymmetrically, creating an alternative dimer interface which coincides with the possible tetramer interface that connects the two canonical dimers. Quaternary state of alternative dimer mimics a closed-state structure in which two recognition helices are distanced at ~ 35 Å and ligand binding pockets are inaccessible. Results of biophysical studies indicate that Fad35R has the propensity to oligomerize in solution in the presence of tetracycline. We present the first structure of a FadR homologue from mycobacterium and the structure reveals DNA and ligand binding features of Fad35R and also provides a view on alternative quaternary states that mimic open and closed forms of the regulator.

Citation

Singh AK, Manjasetty BA, GL B, Koul S, Kaushik A, et al. (2015)
Crystal Structure of Fad35R from Mycobacterium tuberculosis H37Rv in the Apo-State.
PLoS ONE 10(5): e0124333. doi:10.1371/journal.pone.0124333

molecule visualisation
Diffraction images (resolution rings shown)
poster
Data collection and refinement statistics
molecule visualisation
Crystal packing representations of canonical and alternative dimers
Beamline BM14

BM14 was bending magnet 14 beamline at the ESRF, optimised for exploiting anomalous scattering methods MAD/SAD in macromolecular structure determination.