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3k93

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

    Title Crystal structure of phage related exonuclease (YP_719632.1) from HAEMOPHILUS SOMNUS 129PT at 2.15 A resolution. To be published
    Site JCSG
    PDB Id 3k93 Target Id 392534
    Molecular Characteristics
    Source Haemophilus somnus 129pt
    Alias Ids TPS27755,YP_719632.1, 325834 Molecular Weight 25559.84 Da.
    Residues 222 Isoelectric Point 6.01
    Sequence mnnlyhlkvrcsslhkiigepkskadkeagkltdtaksavremakfdlfgynafegnkytqkgneleeq aiklsgvtrglalkknterrenefitgecdiyvpsrkliidtkcswdigshpfftdeaqekakkagydi qmqgymwlwdcdqaqidfvlfptplnlisaydsdfklidlveqipqirrittviiqrdnelidkikerv saaqkyydqlisems
      BLAST   FFAS

    Structure Determination
    Method XRAY Chains 1
    Resolution (Å) 2.15 Rfree 0.212
    Matthews' coefficent 3.03 Rfactor 0.172
    Waters 122 Solvent Content 59.41

    Ligand Information
    Ligands
    Metals

    Jmol

     
    Google Scholar output for 3k93
    1. Structural and functional insight into the mechanism of an alkaline exonuclease from Laribacter hongkongensis
    W Yang, W Chen, H Wang, JWS Ho - Nucleic Acids , 2011 - Oxford Univ Press
     

    Protein Summary

    Protein HS_1420 (JCSG target ID 392534, JCSG target accession code FR14724A , GenBank accession code YP_719632.1) is annotated as a hypothetical protein from the 129PT vaccine strain of Haemophilus somnus, a non-motile, gram-negative bacterium that is pathogenic to cattle.  HS_1420 is a representative of a small (~20 known members) divergent branch of a PFAM YqaJ viral recombinase family (PF09588), present in proteobacteria from the Pasteurellaceae (Haemophilus, Actinobacillus) and Neisseriaceae families, including many human and animal pathogens.  Although the PFAM match to the YqaJ family has low significance (e.value of 0.15), it is supported by strong matches from more sensitive recognition programs (e.g., FFAS and HPRED) as well as PSI-BLAST analysis. This classification is confirmed by structural analysis (see below). Proteins from this family form two-component recombinase pairs (best known example is RecE/T from the Rac prophage of E. coli), which is consistent with the genomic neighborhood of HS_1420.

    The structure of HS_1420 (residues 1-222), solved by the Se-Methionine multi-wavelength anomalous dispersion (MAD) method to a resolution of 2.15 Angstroms, adopts an alpha-beta fold that can be classified in SCOP as belonging to the restriction endonuclease-like superfamily of folds (Figure 1).

    Figure 1.  Structure of a monomer of HS_1420 gradiently colored from the N- (blue) to the C-terminus (red).  Sodium ion is indicated as a green sphere, and chloride ions are represented as magenta spheres.

    FR14724A-gradient.png

     

    Both crystal packing and PISA analyses suggest that the biologically functional form of HS_1420 is a trimer, forming a toroid as shown in Figure 2ab.  A wide tapered channel is formed between the three subunits, measuring ~30 Angstroms at one end and ~15 Angstroms at the other.

    Figure 2.  Quaternary structure of HS_1420.  (a) view along the 3-fold axis and toward the wider end of the channel that is formed in the center of the trimer.  (b) side view, rotated by 90 degrees counterclockwise about the vertical axis from (a). The wider end of the channel is on the right side.  Sodium ions are represented as green spheres.

    (a)                                                                                                        (b)

    FR14724A-trimer1.png  FR14724A-trimer2.png

     

    Comparison with other Structures:

    A Dali search of HS_1420 resulted in several close structural neighbor matches, the best among them being 1avq (a DNA exonuclease from bacteriophage-lambda, [Ref]), 3h4r (DNA exonuclease RecE from E. coli, [Ref]), and 2w45 (a nuclease from Epstein-Barr virus (EBV), [Ref]) (Table 1).

     

    Table 1.  Structural neighbors of HS_1420 as assessed by Dali.

    N PDB Z-score RMSD LALI NRES %ID TITLE
    1 1avq 17.9 2.4 184 228 18 LAMBDA EXONUCLEASE [Ref]
    2 3h4r 9.6 3.2 153 219 10 EXODEOXYRIBONUCLEASE 8 [Ref]
    3 2w4b 9.4 3.2 168 455 12 ALKALINE EXONUCLEASE [Ref]
    4 2w45 9.4 3.2 165 437 13 ALKALINE EXONUCLEASE [Ref]
    5 1w36 7.1 3.7 140 1158 14 DNA HAIRPIN
    6 1gef 5.0 3.2 92 116 14 HOLLIDAY JUNCTION RESOLVASE

     

    A comparison of HS_1420 with 1avq, 3h4r, and 2w45 reveals that HS_1420 is most similar to 1avq.  Although the cores of all of these structures share what is termed an "restriction endonuclease-like" fold (Figure 3), 3h4r and 2w45 are parts of larger proteins and have different quaternary structures (tetramer and monomer, respectively) than either HS_1420 or 1avq (both trimers) (Figures 4ab).

    Figure 3.  Superpositon of HS_1420 (yellow) with 1avq (blue), 3h4r (red), and 2w45 (green) reveals a similar core restriction endonuclase-like fold.  Sodium ion of HS_1420 is represented as a green sphere.

    FR14724A-1avq-3h4r-2w45-superpose.png

    Figure 4. HS_1420 is most similar to lambda-exonuclease (1avq) in terms of both its tertiary and quaternary structures.  Superposition of the (a) monomers and (b) trimers of HS_1420 (yellow) and 1avq (blue).  Sodium ion of HS_1420 is represented as a green sphere.

    (a)                                                                                       (b)

    FR14724A-1avq-superpose.png  FR14724A-1avq-superpose-trimer.png

     

    Active Site:

    1avq, as well the other top structural neighbors listed above (i.e., 3h4r and 2w45), are DNA nucleases that are Mg+2 -dependent enzymes.  Heavy metal soaks with Mn+2 or Ba+2 revealed where the active site Mg+2 would be bound in these structures.

    In the case of HS_1420, a sodium ion has been modeled at the corresponding site (Figure 5).  A sodium, rather than a magnesium ion, was modeled in HS_1420 based on the presence of NaCl and the absence of Mg+2 in the crystallization condition, and also the fact that the observed metal-ligand (X-O) distances in the structure are closer to those of Na-O than those of Mg-O.

    The active site residues are well-conserved between HS_1420 and 1avq.  For example, Asp-99, Asp-110, Lys-112, and the carbonyl oxygen of Thr-111 correspond to active site residues Asp-119, Glu-129, Lys-131, and the carbonyl oxygen of Leu-130 in 1avq.  In HS_1420, three of these residues (Asp-99, Asp-110, and Thr-111), in conjunction with three waters, octahedrally coordinate the sodium ion.

    Figure 5.  Superposition of the active sites of HS_1420 (yellow) and 1avq (blue).  Active site residues are represented as sticks, sodium ion of HS_1420 is indicated by a green sphere, and waters are represented as red spheres.  Sodium-ligand distances are labeled.

    FR14724A-1avq-superpose-activesite.png

     

    Aside from the residues immediately surrounding the sodium, a sequence alignment of HS_1420 with its top ten BLAST hits shows that a number of other residues in the active site region are also highly conserved (Figure 6).

    Figure 6.  Residue conservation mapped onto the structure of HS_1420.  Residues that are the least and most conserved are in blue and red, respectively.

    FR14724A-consurf.png

    consurf_legend.png

     

    Electrostatic Potential Surface:

    If HS_1420 is indeed a DNA nuclease, it may be expected that the channel may have a more positive potential.  An electrostatic potential surface view of HS_1420 (Figure 7) reveals that the central portion of the channel is rather evenly charged, but that the residues surrounding the wider opening of the channel as well as those in the active site area appear to be more positively charged (Figure 7).

    Figure 7.  Electrostatic potential surface of HS_1420 trimer.

    FR14724A-trimer-emap.png

     

    The similarity in structure and active sites between HS_1420 and lambda-exonuclease (1avq) strongly suggests that HS_1420 may also be a DNA exonuclease.  If so, HS_1420 may interact with DNA in a similar fashion as proposed for 1avq.  In this proposed model, dsDNA enters through the wider end of the channel, becomes unwound in the channel, 5' end nucleotides are cleaved at the active site(s), and the 3' strand exits through the narrower opening of the channel (Figure 8).

    Figure 8.  Proposed model of interaction between lambda-exonuclease (1avq) and DNA.  Adapted from [Ref].

    1avq-model.png

    Ligand Summary

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