NMR structure of YcgL, a conserved protein from <i>Escherichia coli</i> representing the DUF709 family, with a novel α/β/α sandwich fold

YcgL represents a conserved class of small proteins widespread in gammaproteobacteria. This group of proteins has no significant sequence similarity with any other protein family and contains a domain of unknown function, DUF709.1, 2 The 108-residue protein YcgL (gi: 26107893) from uropathogenic Escherichia coli3 was selected for structural studies in the context of the research program of the Montreal-Kingston Bacterial Structural Genomics Initiative (BSGI; http://euler.bri.nrc.ca/brimsg/bsgi.html). The ycgL gene was amplified by PCR from Escherichia coli CFT073 genomic DNA and was cloned into a pET15b vector derivative (Amersham Biosciences) to obtain a TEV cleavable N-terminal His8-tagged fusion protein. The expression of ycgL was carried out in LB medium or in minimal M9 medium containing [15N]NH4Cl and [13C] glucose (Cambridge Isotopes Laboratory, Andover, MA). Protein expression was induced by 0.1 mM isopropyl-β-D-thiogalactopyranoside (IPTG). Purification was carried out by affinity chromatography using Ni2+-loaded chelating Sepharose (Amersham Pharmacia Biotech) by standard protocols. For NMR experiments the tag was cleaved using TEV protease. NMR samples were prepared in 50 mM phosphate buffer (pH 6.8), 150 mM NaCl, 15 mM DTT, and 0.02% (w/v) sodium azide. NMR data were collected using Bruker Avance500 spectrometer equipped with a z-gradient cryoprobe and Varian INOVA 500. All experiments were performed at 308.0 K using 1.5 to 2.5 mM samples. Sequence-specific backbone and side chain chemical shifts assignments were obtained from 1H-15N HSQC, 1H-13C HSQC, 2D TOCSY, HNCA, HNCACB, CBCA (CO)NH, HNCO, HN(CA)CO, HNHA, HBHA(CO)NH, 3D-HCCH-TOCSY, 3D 1H-15N TOCSY-HMQC, and 3D 1H-13C NOESY-HSQC experiments. DSS was used as an internal standard for 1H chemical shifts and 13C and 15N were calibrated assuming γ ratios 15N/1H = 0.101329118 and 13C/1H = 0.251449530.4 NMR spectra were processed with XWINNMR (Bruker Biospin) and NMR-Pipe5 software and analyzed using the programs CARA6 and XEASY.7 NMR signal assignments were obtained for all but 6 residues: Met1, Ser9, Lys61, Pro85, Pro86, Pro87, and Pro81. NOE-restraints were collected from 3D 1H-15N NOESY-HMQC, 3D 1H-13C NOESY-HSQC (in D2O) and 2D homonuclear-NOESY (in H2O and D2O) experiments. The ϕ and ψ torsion angles were derived from Cα, Cβ, C′, Hα, and N chemical shifts using TALOS8 program. The YcgL structures were calculated with CANDID/CYANA 2.19, 10 using 200 starting conformers. NOEASSIGN/CYANA 2.1 protocol was applied to calibrate and assign NOE cross-peaks. NOE upper limit distances were used together with 56 pairs of dihedral angles and 19 hydrogen bonds derived from a 1H-15N HSQC deuterium exchange experiment. The structure was refined using Xplor-NIH.11 The average number of NOEs per residues is 22 for the structured region. Structural statistics are shown in Table I. The programs SYBYL (6.9 version; Tripos, St. Louis, MO), PROCHECK-NMR12 and MOLMOL13 were used for NOEs violations interpretation and secondary structure definition. The NMR structure of YcgL was determined using the program Cyana 2.1. The 40 lowest energy structures were further refined in Xplor-NIH. An ensemble of the 20 lowest energy conformers is shown in Fig. 1A and the structure calculation summary is given in Table I. Three-dimensional NMR solution structure of YcgL. (A) Stereo view of the superposition of the final 20 energy-minimized structures and ribbon diagram (in cyan). (B) Ribbon presentation showing the topology of YcgL. β-strands and α-helices are shown in yellow and red, respectively (The unstructured parts comprising residues: Met3 to Lys10 and Lys93 to Lys108 are not shown). (C) Sequence alignment in ClustalW14 of YcgL from different bacteria: (AAN80092) Escherichia coli CFT073, (NP_460769) Salmonella typhimurium LT2, (CAG75270) Erwinia carotovora subsp. atroseptica SCRI1043, (AAS62141) Yersinia pestis biovar Medievalis str. 91001, (BAE74612) Sodalis glossinidius str. “morsitans,” (CAE14432) Photorhabdus luminescens subsp. laumondii TTO1, (ZP_01235889) Vibrio angustum S14, (ZP_01162134) Photobacterium sp. SKA34, (AAZ25145) Colwellia psychrerythraea 34H, (ZP_01216678) Psychromonas sp. CNPT3, (CAI89545) Pseudoalteromonas haloplanktis TAC125, (ZP_00838565) Shewanella sp. PV-4, (ZP_00775422) Pseudoalteromonas atlantica T6c, (ZP_01109765) Alteromonas macleodii ‘Deep ecotype’, (ZP_01043739) Idiomarina baltica OS145, (AAC23096) Haemophilus influenzae, (AAG04684) Pseudomonas aeruginosa PAO1, (YP_449457) Xanthomonas oryzae pv. oryzae MAFF 311018. The amino acids 1 to 11 and 96 to 108 [E.coli YcgL] were omitted because there is no sequence conservation in these regions. The secondary structure of YcgL is shown at the bottom of the sequence alignment. The PyMOL (DeLano Scientific LLC) and SYBYL (Sybyl 6.9 software (Tripos, St. Louis, MO)) programs were used for A and B panels' design. The YcgL NMR structure contains two α-helices and four β-strands in the sequential arrangement β1-β2-α1-β3-α2-β4 as a 3-layer (α/β/α) sandwich. The β-sheet comprises residues Cys14 to Ser19 (β1), Tyr26 to Val29 (β2), Pro49 to Leu55 (β3) and Tyr80 to Glu83 (β4) adopting an anti-parallel ↓3↑1↓2↑4 topology. The helices α1 (residues Pro39 to Gly47) and α2 (residues Asp67 to Gln78) are arranged antiparallel to each other on one face of the β-sheet (Fig. 1B). The β4 strand has a bulge at Ala51. Two loops between β2-α1 and β3-α2 are long with 9 and 12 residues, respectively. The region Arg60 to Val66 of the second loop has very few NOEs, suggesting its mobility. The YcgL surface does not reveal any obvious cavity or specific active site. Analysis of the sequence conservation for homologous proteins from the 18 most diverse bacteria reveals high conservation in the structured part of the protein (Fig. 1C). Nearly all of the conserved residues contribute to formation of the observed fold. A conserved hydrophobic core stabilizes the globular fold of YcgL. Both connections between α-helices and β-strands (α1-β3 and α2-β4) are short and include two conserved glycines (Gly47 and Gly79). In the loop between strands β1 and β2 there are two conserved residues, Ser19 and Arg22 (or Lys), with potential functional implications, e.g. for partner binding. The amino acids in the fourth β-strand are very conserved and this strand is followed by a proline-rich region in most related sequences. The β1-β2 loop region and C-terminal proline-rich region are close in space and therefore may jointly participate in partner binding. We analyzed the YcgL protein sequence using residues Phe13 to Leu94 with RSP-BLAST15 and PFAM2 and identified this as a domain of unknown function (DUF709, COG3100). The BLAST alignment shows very high conservation for the sequences in the DUF709 family and low score for other proteins. This domain definition for E. coli YcgL is consistent with the structured region determined by NMR (Fig. 1C). Three-dimensional structure similarity analysis using DALI16 program gave two hits with a low Z score at 1.9, Argonaut N-terminal mRNA interacting domain (pdb code 1U04). and a catalytic domain of homing endonuclease (pdb code 1LN0). YcgL and N-terminal domain from Argonaut overlap all β-strands from β-sheet and one helix (α2 from YcgL). Both proteins have quite different topology from YcgL. Two hits in VAST17 carboxypeptidase inhibitor (LCI) (pdb codes 1DTD and 2ABZ) and folding chaperone SurA (pdb code 1M5Y) have low log(P) scores of 1.69 and 1.39, respectively. YcgL and SurA structure superposition includes only two β-strands and show low 3D similarity. LCI has similar overall topology for the β-sheet, but contains one additional β-strand, only one helix and four disulfides bridges. Interestingly, the conserved β1-β2 loop and proline-rich regions overlap with the interaction surface of LCI and suggests a putative binding site for a protease. Low structural similarity between YcgL and other proteins from databases suggest a novel α/β/α sandwich fold for YcgL protein. The atomic coordinates, signal assignments and NMR constrains have been deposited in the Protein Data Bank with accession code 2H7A. The authors thank Dr. Traian Sulea, for assistance in bioinformatics studies and very useful discussions, Dr. Allan Matte for careful reading of the manuscript, and Jing Cheng for her help in protein purification. We thank Dr. Tara Sprules for the 2D-13C HSQC, NHCACB, and CBCA(CO)NH experiments recorded at the Québec/Eastern Canada High Field NMR Facility.

Récupéré en direct depuis OpenAlex et désinversé. Les résumés ne sont pas conservés dans cette base de données : les index inversés représentent 8,6 Go des 9,3 Go de texte de la base, et le serveur dispose de 13 Go libres.