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2h1t

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

    Title The structure of the first representative of Pfam family PF06475 reveals a new fold with possible involvement in glycolipid metabolism. Acta Crystallogr.,Sect.F 66 1211-1217 2010
    Site JCSG
    PDB Id 2h1t Target Id 361385
    Molecular Characteristics
    Source Pseudomonas aeruginosa pao1
    Alias Ids TPS1469,NP_250684.1, PF06475, 90808 Molecular Weight 21559.91 Da.
    Residues 187 Isoelectric Point 4.94
    Sequence msrdrlytwaglwrspssswealrleddqaesqlrapdersglpyqldyrlrwdadwhlreavfhvese tgvrklhlladgrghwqdgdgealpafdgcldidiwpspftntfpirrlgladgqraeiralyieapal eprsmrqaytrldashylyenlegsafkavllvdeqglvidypglfqrl
      BLAST   FFAS

    Structure Determination
    Method XRAY Chains 2
    Resolution (Å) 1.80 Rfree 0.213
    Matthews' coefficent 2.46 Rfactor 0.17
    Waters 367 Solvent Content 50.05

    Ligand Information
    Ligands
    Metals

    Jmol

     
    Google Scholar output for 2h1t
    1. 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
     
    2. Structural classification of proteins and structural genomics: new insights into protein folding and evolution
    A Andreeva, AG Murzin - Acta Crystallographica Section F: Structural , 2010 - scripts.iucr.org
     
    3. The structure of the first representative of Pfam family PF06475 reveals a new fold with possible involvement in glycolipid metabolism
    C Bakolitsa, A Kumar, D McMullan - Section F: Structural , 2009 - scripts.iucr.org
     

    Protein Summary

    The PA1994 gene from Pseudomonas aeruginosa endoces the NP_250684 amino acid sequence belonging to the protein family PFAM:PF06475 (DUF1089), an uncharacterized group of conserved bacterial proteins (COG:COG3554). The PA1994 structure (2h1t) provides the first structural representative of this family and suggests a potential binding site and an involvement in glycolipid metabolism likely induced under conditions of host pathogen interactions or bacterial cell-wall stress.

     

    SCOP classifies 2h1t as an all-beta protein that adopts a novel fold (spiral β-roll) composed of a 15-stranded β-sheet wrapped around a single helix. In the crystal structure, part of the 2h1t fold is formed by  β-strand-swapping between two monomers (Fig. 1). This exchange results in an extensive dimerization interface with ~3000 Å^2 buried per monomer.


    2h1tdimer.png

        

    Figure 1. Ribbon diagram of the 2h1t dimer. Monomers are depicted in blue and magenta. An exchangeable β-strand region, involving the first two N-terminal β-strands, is swapped between the two monomers.


    A search with FATCAT shows a structural similarity of 2h1t with several membrane proteins, including the outer membrane protein NspA from Neisseria meningitidis (PDB:1p4t ) and OmpG, a porin found in Gram negative bacteria (PDB:2f1c ).  The strongest similarity occurs with the lipoprotein localization factors A and B (LolA, LolB) from Escherichia coli, highly conserved bacterial proteins implicated in lipoprotein sorting and membrane localization ([Ref]), with an RMSD of 3.1 Å and a sequence identity of 5% over 104 residues for LolA (Fig. 2). LolA and PA1994 share the same topology over 11 β-strands and the central helix. The main differences are the LolA C-terminal 310-helix and β-strand, structural elements absent in PA1994 and LolB that have been involved in the specific membrane localization of lipoproteins by LolA ([Ref]), and an additional N-terminal helix in LolA located at the bottom of the β-barrel (Fig. 2). The two N-terminal β strands, that are swapped in the PA1994 dimer, are replaced in LolA by a loop and β-strand (Fig. 2).

        

    2h1t1iwl.png

        

    Figure 2. Stero ribbon diagram showing the structural superposition of PA1994 (magenta, pdb id: 2h1t) and LolA (gray, pdb id: 1iwl). Visible on the left, the LolA C-terminal 3-10 helix and beta strand implicated in specific membrane localization of lipoproteins.

        

    An analysis of 2h1t structure using the CastP server reveals a deep pocket (15 Å x 6 Å x 7 Å) enclosed mainly by helix H2 and the preceding strand (β7), with additional contributions made by strands β10-β12 and the loop between strands β14-β15. The pocket is lined with conserved hydrophilic residues (Ser107, Thr110, Asn111, Thr112, Gln145) and the hydroxyl group of the invariant Tyr147 (Fig. 3), and contains an acidic pocket formed by two invariant aspartates (Asp101, Asp103) (Fig. 4). The pocket is located in the vicinity of a cavity that in LolA has been shown to bind to lipids ([Ref]). However, in LolA the binding pocket is hydrophobic whereas in PA1994 is acidic suggesting a hydrophilic ligand.  In addition, the entrance to the pocket forms a long and narrow groove (20 Å x 7 Å) composed of strictly or highly conserved hydrophobic residues (Ile102, Pro106, Pro108, Phe165, Leu170, Ile178) and also involves the dimerization interface (Trp13), suggesting a hydrophobic component to the ligand and the likely requirement of dimerization for binding. 

        

    HMM          tvstavvrWRelWdgeGlEhllLrqsDdglgkrirAegVvigerggtNPayglrYrlecDagWHLRtrrfrVdvtlGERgReLhlasDRRGdGhWrdtDldGeplpaldGclDiDLgfTPFTNtLPIRRLgLsergesaeipvaYvefPaqiDGDlepdsaeQsYTCLDegRlYlYearsGsaFdaeLeVDedGLVvDYPgLfqRl
    MATCH        ++++++++W++lW+++++++++Lr++Dd++++++rA+    +er+g+  +y+l+Yrl++Da+WHLR+++f+V++++G  +R+Lhl++D  G+GhW+d+  dGe+lpa+dGclDiD++++PFTNt+PIRRLgL+ +g++aei+++Y+e+Pa     lep+s++Q+YT+LD+++ YlYe+++GsaF+a+L+VDe+GLV+DYPgLfqRl   
    PA1994       MSRDRLYTWAGLWRSPSSSWEALRLEDDQAESQLRAP----DERSGL--PYQLDYRLRWDADWHLREAVFHVESETG--VRKLHLLAD--GRGHWQDG--DGEALPAFDGCLDIDIWPSPFTNTFPIRRLGLA-DGQRAEIRALYIEAPA-----LEPRSMRQAYTRLDASH-YLYENLEGSAFKAVLLVDEQGLVIDYPGLFQRL    187  

        

    Figure 3.  DUF1089 protein family signature (Pfam) aligned with PA1994. The alignment shows conservation of N-terminal tryptophans involved in stabilizing the dimerization interface as well as of residues implicated in formation of the PA1994 pocket.

        

    A search against a database of cognate binding sites using IsoCleft ([Ref]) identified shared features between the PA1994 pocket and the binding sites of proteins implicated in bacterial cell-wall biosynthesis, with an alanine racemase from Pseudomonas aeruginosa (PDB id: 1rcq) and a hyaluronate lyase from Streptococcus pneumoniae (PDB id: 1loh) as the top hits. Additional similarities implicate the binding of sugars, with mutarotase (PDB id: 1so0) and meso-2,3-butanediol dehydrogenase (PDB id: 1geg) as the closest hits, and an inorganic pyrophosphatase (PDB id: 1wpm). The hydrophobic groove along the 2h1t pocket entrance and dimerization interface gave, as top hit in IsoCleft, a lipase binding site from Candida rugosa (PDB id: 1lpn) ([Ref]). 

        

    Taken together, these structural and chemical similarities strongly support a role for PA1996 and the DUF1089 family in glycolipid metabolism. Glycolipids act as key immunomodulatory molecules in host-pathogen interactions ([Ref]) and lipases have been known to act as virulence factors ([Ref]). Glycophospholipids implicated in the synthesis of complex cell-wall structures that help a pathogen evade a host’s immune system defenses, have been suggested to bind to similar-sized acidic pockets as the one described for PA1996 ([Ref]). Structural analysis additionally suggests that a dimer is likely the biologically relevant oligomerization state of PA1996, and that substrate binding might induce large-scale conformational changes, as is the case with the lipid-binding proteins that share structural similarities with PA1996 ([Ref],[Ref],[Ref]).

        

    Fig3.png

     

    Figure 4. Electrostatic potential surface representation of 2h1t. Red indicates acidic regions. The cross-section occurs along the central helix and the preceding β-strand. The three strands following the helix form the second half of the cavity (not shown). Acidic residues conserved within the DUF1089 family are indicated.

        

    In addition to their role in pathogenicity, bacterial cell wall glycolipids are modified in response to variations in temperature, pH and other environmental stressors ([Ref]) with changes affecting both the lipid and sugar composition of the membrane ([Ref],[Ref]). The genome context (http://string.embl.de) of DUF1089 family members additionally supports a role in glycolipid biosynthesis, likely induced under conditions of cell-wall stress or host-pathogen interactions. PA1994 shows a high degree of confidence (0.8) in a predicted functional association with a peptidyl prolyl cis-trans isomerase (PA1996), an enzyme that functions as a chaperone and it is up-regulated under conditions of cell-wall stress ([Ref]). Similarly, R02764, a DUF1089 homologue from Sinorhizobium meliloti, is predicted to be functionally linked to a glyceraldehyde 3-phosphate dehydrogenase (R02763, a normally cytosolic enzyme involved in energy metabolism that shows pH-dependent association with bacterial cell walls ([Ref]) where it becomes involved in host-pathogen interactions ([Ref])), a transketolase (R02762, an enzyme implicated in lippopolysaccharide metabolism ([Ref])) and a taurine uptake ABC transporter (RB0965, taurine is a constituent of bacterial cell walls that has been implicated in membrane stabilization and recovery from osmotic shock ([Ref])). MT3862, a DUF1089 homologue from Mycobacterium tuberculosis, shows a high degree of confidence in a predicted functional association with two osmoprotectant proteins (MT3863, MT3864) implicated in glycine betaine-dependent transport. In addition to its role in maintaining membrane fluidity, glycine betaine acts as a chemical chaperone ([Ref]) stabilizing proteins under conditions of environmental stress.

    Ligand Summary

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    Files (3)

    FileSizeDateAttached by 
     2h1t1iwl.png
    No description
    87.89 kB03:22, 3 Jul 2008tinabActions
    2h1tdimer.png
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    693.89 kB20:28, 2 Jul 2008tinabActions
     Fig3.png
    DUF1089 conserved acidic pocket
    49.83 kB00:17, 9 Jul 2008tinabActions
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