CalE6 from is a (pyridoxal 5 phosphate) PLP-dependent methionine (putative cysteine desulfurase), (putative selenocysteine lyase/cysteine desulfurase), and (methionine lyase activity. complicated structure subsequently reported by Track (PDB code: 4U1T).34 Despite little structural difference between the two ligand bound forms, all four subunits consistently showed nontrivial local conformational changes including 17 side-chain rotation of the active site residue Tyr 100, a highly conserved residue structurally stacking with the pyridine ring of the cofactor in PLP-dependent enzymes. We further simulated the dynamics and sampled the conformational space of this enzyme using both coarse-grain elastic network models and ensemble refinement methods based on the CalE6-MES complex structure. We exhibited that computational analysis, complementary to X-ray crystallography not only consistently recognized the global distribution of protein mobility but also allowed deriving multiple-conformer ensemble models from diffraction data to properly represent the local dynamics including the rotation mode of active site Tyr 100 of CalE6, as probed by the latter method. II.?MATERIALS AND METHODS A. Cloning and protein expression Cloning and expression protocols of CalE6 followed the standard high throughput procedures of Midwest Center for Structural Genomics, the details of which are also available at TargetTrack database (www.sbkb.org/tt/) under Project Target ID APC109014, as a Protein Structural Initiative target. A brief summary was provided here. The full length gene from (gi: 22255867) was amplified from your genomic DNA using forward primer 5-TACTTCCAATCCAATGCCGTGAGCGGTATGCGCTTCGAC-3 and reverse primer 5-TTATCCACTTCCAATGTTAGGTGCCGCCCGCCAG-3. The PCR product was cloned into vector pMCSG73 according to the ligation-independent process and transformed into the BL21(DE3)-Platinum strain (Stratagene). The vector pMCSG73 is derived from vector pMCSG53 and contains tRNA genes covering rare codons Arg (AGG/AGA) and Ile (AUA).36 DNA sequencing identified a mutation corresponding to D7G variation at the N-terminus, which was subsequently confirmed to show no effect on the overall or remote active site structure of the protein. Supplementary Physique S1 shows the local loss of a salt bridge with Arg 253 from a neighbor subunit and concomitant side-chain reorientation of Arg 253 due to D7G variance without affecting the overall structure.61 Selenomethionine labeled protein was overexpressed from BL21 (DE3) culture grown in M9 minimal media supplemented with inhibitory amino acid cocktail and Se-Met under induction condition of 0.5?mM IPTG at 18?C overnight. The fusion product contains a TVMV-cleavable N-terminal NusA tag followed by a TEV-cleavable N-terminal 6xHis tag (NusA-ETVRFQ/S-HHHHHH-WSHPQFEK-ENLYFQ/SNA-TARGET). After cell lysis by sonication, the Se-Met labeled protein was purified by Ni-NTA affinity chromatography using AKTAxpress system (GE Health Life Sciences, USA), TEV protease cleavage, followed by an additional subtractive IMAC 414864-00-9 supplier step to remove the protease, uncut protein, and affinity tag. The purified untagged protein was concentrated to 11?mg/ml with Amicon Ultra-15 centrifugal concentrators (Millipore, Bedford, MA, USA) and stored in 20?mM HEPES pH 8.0, 250?mM NaCl, 2?mM dithiothreitol, and 1?mM PLP at ?80?C. B. Protein crystallization Several commercially available crystallization screens (MCSG-1C4, Microlytic, Inc. MA, USA)37 were used, which led to identification of multiple CalE6 crystallization conditions. The best diffracting CalE6 crystal was obtained using the sitting drop vapor diffusion method 414864-00-9 supplier by mixing 0.4?atoms in all the superposed models. The larger the average scaled distance, the higher relative mobility of the residue in the ensemble models. III.?RESULTS AND DISCUSSION A. The crystal structure of CalE6 and ligand induced rotation of Tyr 100 The crystal structure of CalE6 was decided and processed to a resolution of 2.1??. Two homotetramers were found in the asymmetric unit displaying essentially the same conformational state with overall CRMSD of 0.143 over 1509 residues of four chains (ACD vs. ECF). The buried surface area of individual tetramer is usually 24,505??325??2. Each tetramer harbors four molecules of PLP, each bound in a cleft created at C-2 symmetry-related dimer interfaces (Physique 2(a)). Within the dimer, one subunit provides Lys 197 as the Schiff-base anchor for PLP and forms multiple non-covalent interactions with the cofactor from residues Ser 75, Gln 77, Tyr 100, Glu 143, Asp 172, Thr 174, Ser 194, Thr 196 (Physique 2(b)). The adjacent subunit provides additional charge-charge and hydrogen-bonding interactions with the phosphate group of PLP from residues Arg 48 and Tyr 46 located on an extended loop (residues 14C51). The same loop also forms quaternary interactions with symmetry-related loops from a distant subunit to hold together the tetramer (Physique 2(b)). A solvent molecule 2-(N-morpholino)ethanesulfonic acid (MES) was recognized in each putative substrate site based on obvious electron density (Physique 2(c)), with Col4a6 the sulfonic group of MES located at an comparative position to the (PDB access 3AEM) (Physique 2(c)).51 FIG. 2. CalE6 overall structure and the 414864-00-9 supplier active site. (a) 414864-00-9 supplier Tetrameric structure of CalE6 holoenzyme. Four subunit chains were shown as.