Crystal structure of Middle East respiratory syndrome coronavirus helicase.

Middle East respiratory syndrome coronavirus (MERS-CoV) remains a threat to public health worldwide; however, effective vaccine or drug against CoVs remains unavailable. CoV helicase is one of the three evolutionary most conserved proteins in nidoviruses, thus making it an important target for drug development. We report here the first structure of full-length coronavirus helicase, MERS-CoV nsp13. MERS-CoV helicase has multiple domains, including an N-terminal Cys/His rich domain (CH) with three zinc atoms, a beta-barrel domain and a C-terminal SF1 helicase core with two RecA-like subdomains. Our structural analyses show that while the domain organization of nsp13 is conserved throughout nidoviruses, the individual domains of nsp13 are closely related to the equivalent eukaryotic domains of Upf1 helicases. The most distinctive feature differentiating CoV helicases from eukaryotic Upf1 helicases is the interaction between CH domain and helicase core

Selectively Disrupting m6^{6}A-Dependent Protein-RNA Interactions with Fragments

We report a crystallographic analysis of small-molecule ligands of the human YTHDC1 domain that recognizes N6-methylated adenine (m$^{6}$A) in RNA. The 30 binders are fragments (molecular weight < 300 g mol$^{-1}$) that represent 10 different chemotypes identified by virtual screening. Despite the structural disorder of the binding site loop (residues 429-439), most of the 30 fragments emulate the two main interactions of the -NHCH$_{3}$ group of m$^{6}$A. These interactions are the hydrogen bond to the backbone carbonyl of Ser378 and the van der Waals contacts with the tryptophan cage. Different chemical groups are involved in the conserved binding motifs. Some of the fragments show favorable ligand efficiency for YTHDC1 and selectivity against other m$^{6}$A reader domains. The structural information is useful for the design of modulators of m$^{6}$A recognition by YTHDC1

Jungfraujoch: hardware-accelerated data-acquisition system for kilohertz pixel-array X-ray detectors

The JUNGFRAU 4-megapixel (4M) charge-integrating pixel-array detector, when operated at a full 2 kHz frame rate, streams data at a rate of 17 GB s−1. To operate this detector for macromolecular crystallography beamlines, a data-acquisition system called Jungfraujoch was developed. The system, running on a single server with field-programmable gate arrays and general-purpose graphics processing units, is capable of handling data produced by the JUNGFRAU 4M detector, including conversion of raw pixel readout to photon counts, compression and on-the-fly spot finding. It was also demonstrated that 30 GB s−1 can be handled in performance tests, indicating that the operation of even larger and faster detectors will be achievable in the future. The source code is available from a public repository

Overall structure of MERS-CoV nsp13.

<p>(A) Ribbon model of MERS-CoV nsp13 containing CH (orange), Stalk (magenta), 1B (blue), RecA1 (red) and RecA2 (green) domains. (B) Schematic diagram of the domain organization of MERS-CoV nsp13.</p

Key structural features of MERS-CoV nsp13.

<p>(A) left, ribbon model of the CH and Stalk domains of MERS-CoV nsp13 colored by secondary structural elements; right, 2-D topology graph of the CH domain. (B) ribbon model of N-terminal Ring module, and (C) C-terminal Ring module of the CH domain. His/Cys residues participating in zinc coordination are highlighted in yellow. (D) Surface representation of CH domain (orange) of MERS-CoV nsp13, two hydrophobic pockets equivalent to protein interaction interfaces on Upf1 are highlighted in green (pocket 1) and cyan (pocket 2). (E) The ATPase active site between RecA1 and RecA2 domains. The conserved helicase motifs are highlighted with different colors.</p

Pairwise comparison of the isolated CH/ZBD, 1B and helicase core domains of MERS-CoV nsp13, EAV nsp10 and Upf1 helicases.

<p>Pairwise comparison of the isolated CH/ZBD, 1B and helicase core domains of MERS-CoV nsp13, EAV nsp10 and Upf1 helicases.</p

Domain composition of Upf1-related helicases.

<p>Ribbon models of (A) MERS-CoV nsp13, (B) EAV nsp10-DNA PDB ID: 4N0O, (C) Yeast Upf1-RNA PDB ID: 2XZL and (D) Human Upf1 without RNA PDB ID: 2WJV colored by different functional domains. The coloring scheme is indicated on the right.</p

Enzymatic activities of recombinant MERS-CoV nsp13.

<p>MERS-CoV nsp13 carries NTPase activity and RNA duplex unwinding activity. (A) Final purification step of recombinant MERS-CoV nsp13 expressed in insect cells. MERS-CoV nsp13 eluted from Superdex 200 300/10 GL column pre-calibrated with gel filtration standards (thyroglobulin 670 kDa, γ-globulin 158 kDa, ovalbumin 44 kDa and myoglobin 17 kDa and vitamin B12 1,35 kDa). Upper insert, SDS-PAGE analysis of the purified protein. (B) ATPase activity of MERS-CoV nsp13. The velocity of ATP hydrolysis is plotted as the function of ATP concentration. The data was fitted to Michaelis–Menten equation to calculate <i>V</i>_<i>max</i> and <i>K</i>_<i>m</i>. (C) Left, helicase assay shows that MERS-CoV nsp13 can unwind RNA partial duplex with 5’ overhang, but cannot unwind RNA partial duplex with 3’ overhang. RNA strand with HEX label is marked with asterisk. Heat denatured and no enzyme (w/o) controls are indicated. (C) Right, helicase assay showing MERS-CoV nsp13 can utilize different NTPs and dNTP to separate RNA strands with a preference to ATP. The sequence of the RNA substrates is shown at the bottom.</p

Data collection and refinement statistics.

<p>Data collection and refinement statistics.</p