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1vk8

    Table of contents
    1. 1. Protein Summary
    2. 2. Ligand Summary
    3. 3. References

    Title TM0486 from the hyperthermophilic anaerobe Thermotoga maritima is a thiamin-binding protein involved in response of the cell to oxidative conditions. J.Mol.Biol. 400 463-476 2010
    Site JCSG
    PDB Id 1vk8 Target Id 282359
    Molecular Characteristics
    Source Thermotoga maritima msb8
    Alias Ids TPS1207,TM0486, 84919 Molecular Weight 10804.93 Da.
    Residues 94 Isoelectric Point 5.46
    Sequence mpkvtvsikvvpavedgrlhevidraiekisswgmkyevgpsnttvegefeeimdrvkelaryleqfak rfvlqldidykaggitieekvskyr
      BLAST   FFAS

    Structure Determination
    Method XRAY Chains 4
    Resolution (Å) 1.80 Rfree 0.18956
    Matthews' coefficent 1.97 Rfactor 0.14151
    Waters 356 Solvent Content 36.95

    Ligand Information
    Ligands
    Metals

    Jmol

     
    Google Scholar output for 1vk8
    1. The Buccaneer software for automated model building. 1. Tracing protein chains
    K Cowtan - Acta Crystallographica Section D: Biological , 2006 - scripts.iucr.org
     
    2. Decision-making in structure solution using Bayesian estimates of map quality: the PHENIX AutoSol wizard
    TC Terwilliger, PD Adams, RJ Read - Section D: Biological , 2009 - scripts.iucr.org
     
    3. High-throughput protein production for X-ray crystallography and use of size exclusion chromatography to validate or refute computational biological unit predictions
    D McMullan, JM Canaves, K Quijano - Journal of structural and , 2005 - Springer
     
    4. Autoindexing with outlier rejection and identification of superimposed lattices
    NK Sauter, BK Poon - Journal of Applied Crystallography, 2010 - scripts.iucr.org
     
    5. TM0486 from the Hyperthermophilic Anaerobe Thermotoga maritima is a Thiamin-binding Protein Involved in Response of the Cell to Oxidative Conditions
    Z Dermoun, A Foulon, MD Miller, DJ Harrington - Journal of molecular , 2010 - Elsevier
     
    6. Applications of Bioinformatics to Protein Structures: How Protein Structure and Bioinformatics Overlap
    GW Han, C Rife, MR Sawaya - Methods in Molecular Biology, 2009 - Springer
     

    Protein Summary

    The TM0486 gene encodes the protein NP_228296 containing a domain of unknown function DUF77 PF01910.  This protein can be found in archaebacteria, eukaryotes and eubacteria.  The crystal structures of its yeast and archaea orthologs have been determined [Ref] with the authors suggesting that the family likely forms a protein–protein interaction module that may be regulated by the binding of small-molecule ligands (possibly sulfate ions). The genomic neighborhood of TM0486 includes (score 0.9) the TM0485 and TM0483 genes annotated as ABC transporters.

     

    A search with the remote sequence homology server HHPred against the Pfam database, gives a strong prediction (98.4% probability, P-value 2.9E-10) for TM0486 being a YkoF-related protein (PF07615). YkoF binds thiamin and is involved in the hydroxymethyl pyrimidine (HMP) salvage pathway [Ref]. SCOP classifies the 1vk8 structure as adopting a ferredoxin-like fold also known as an ACT domain [Ref]. A search with DALI shows a significant structural similarity with YkoF protein 1sbr (Z-score 7.0, rmsd 2.0 Å over 80 residues, 11% identity). The TM0486 dimer can be superimposed along the YkoF monomer (Fig. 1) with the TM0486 tetramer and YkoF dimer (functional YkoF unit) also being superimposable. This similarity at the quaternary structure level, suggests that YkoF may have arisen through duplication of a DUF77 (or DUF77-like) unit. However, YkoF (PF07615) is only found in bacteria.

     

    1vk8_1s7h(2).png

     

    Fig. 1. Ribbon diagram of YkoF (1sbr, blue) superimposed on a TM0486 dimer (1vk8, green and magenta) indicates that YkoF likely arose through duplication of a DUF77 unit.

     

    The YkoF dimer contains 4 thiamin/HMP (hydroxymethyl pyrimidine) binding sites, two high affinity ones at the N-terminal end and two lower affinity ones at the C-terminal region. If YkoF arose through gene duplication, it is possible that the C-terminal binding site presents a degenerate version of the initial N-terminal region.

     

    The TM0486 tetramer also contains four similarly placed UNL (unknown ligands). Conservation analysis (ConSurf) shows a few strictly conserved residues (Arg70, Lys89, Tyr93) indicative of this being an enzyme. Lys89 and Tyr93 are positioned next to the C-terminal UNLs in the structure (Fig. 2) suggesting that similarly, the ligand could be a thiamin or HMP analog. Genome context also supports this  hypothesis giving a 98% probability of a functional association with TM0484, a putative pyrimidine precursor biosynthesis enzyme, and 61% probability of a functional association with thiC, a thiamin biosynthesis precursor. Other homologs from this family have been solved by structural genomic projects including 2IBO, 1YQH, 2EKY, 2EPI, 1LXJ and 1LXN.

     

    UNL2.png            Thiamin2.png

     

    Fig. 2. Structural superposition of TM0486 (PDB:1VK8, in magenta) with the YkoF protein from Bacillus subtilis (PDB:1SBR, in blue). RMSD is 2Å over 80 residues, 11% identity. The dummy atoms modeled into the unknown ligand (UNL) overlap with the pyrimidine ring of thiamin while the interacting residues (Ile, Ser/Thr, Leu/Val) are also conserved.

     

    Gene inactivation of the DUF77 yeast homolog makes yeast hypersensitive to agents disrupting cell-wall polymers, a result that was interpreted as DUF77 having a role in cell wall formation [Ref]. Based on the conservation of residues involved in oligomerization and the presence of ordered sulfates in the crystal structure, another proposed functional role involved DUF77 acting as a protein-protein interaction module [Ref]. The Thermotoga maritima homolog is upregulated during biofilm formation with the authors suggesting a role in sulfate/sulfonate uptake [Ref].

     

    All of the above observations can be explained by a role of DUF77 in thiamin biosynthesis or salvage. Thiamin derivatives, such as thiamin pyrophosphate, are required for yeast cell wall integrity in the role of co-factors of essential outer wall enzymes such as saccharases and other cell-wall invertases [Ref] while thiamin biosynthesis is strongly upregulated during biofilm formation in E. coli [Ref].

     

    Recently, a new thiamin salvage pathway was identified with a wide distribution among bacteria, archaea and eukaryotes [Ref] and the possibility has been raised that other such pathways remain to be discovered [Ref]. Based on the above and by analogy with YkoF's function in HMP salvage, we propose that DUF77 homologs may be part of such a pathway.

     

    Further investigation:

    Investigate ligand coordination more closely (e.g. TM0486 C-terminal helix is absent in YkoF structure but is present in YkoF sequence - ligand-induced helix-formation?). Do all DUF77 homologs have this helix?

    • Compare residue conservation between the two binding sites (further explore gene duplication hypothesis, i.e. weak, C-terminal YkoF site being a degenerate version of the N-terminal one)
    • Compare residue conservation between the two binding sites (could the weak, C-terminal YkoF site be a degenerate version of the N-terminal one?)
    • Compare functional couplings for YkoF with those found for DUF77 homologs.


    Ligand Summary


    References

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    Files (3)

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     1vk8_1s7h(2).png
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    169.66 kB23:30, 15 Dec 2009tinabActions
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