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

    Title Crystal structure of a putative aminoglycoside phosphotransferase (YP_614837.1) from Silicibacter sp. TM1040 at 2.15 A resolution. To be published
    Site JCSG
    PDB Id 3csv Target Id 387075
    Molecular Characteristics
    Source Silicibacter sp. tm1040
    Alias Ids TPS1774,YP_614837.1, 283092 Molecular Weight 37759.50 Da.
    Residues 332 Isoelectric Point 5.26
    Sequence mtsredeirdflathgyadwnrtplagdassrryqrlrsptgakavlmdwspeeggdtqpfvdlaqylr nldisapeiyaeehargllliedlgdalftevinndpaqemplyraavdllihlhdaqtpelarldpet lsemtrlafseyryailgdaaednrkrfehrfaqilsaqlegdmvfvhrdfhaqnllwlpereglarvg vidfqdaklghraydlvsllqdarrdvpaqveaqmidhyiqatgvdeshfrsayaviavqrnmrilgif arlsqrfgkrhyiefvprvwahferglahpalasaaeeilnalpapapevlerlra
      BLAST   FFAS

    Structure Determination
    Method XRAY Chains 1
    Resolution (Å) 2.15 Rfree 0.228
    Matthews' coefficent 2.48 Rfactor 0.168
    Waters 199 Solvent Content 50.48

    Ligand Information
    Ligands
    Metals

    Jmol

     
    Google Scholar output for 3csv
    1. Aminoglycoside-2 phosphotransferase-IIIa (APH (2)-IIIa) prefers GTP over ATP: Structural templates for nucleotide recognition in the bacterial aminoglycoside-2
    CA Smith, M Toth, H Frase, LJ Byrnes - Journal of Biological , 2012 - ASBMB
     

    Protein Summary

    YP_614837.1 from Silicibacter sp. TM1040 encodes the first solved structure of the chloro subfamily of Choline and Aminoglycoside Kinases (CAK), which are in turn members of the Protein Kinase-Like (PKL) superfamily. CAKs are best known as small molecule kinases, including choline kinases from eukaryotes and eubacteria, and aminoglycoside phosphotransferases, which inactivate aminoglycoside antibiotics by phosphorylation. Aminoglycoside phosphotransferases are a major contributor to antibiotic resistance of drugs that target the 16-S ribosomal subunit. These enzymes are highly promiscuous and can inactivate multiple drugs (Nurizzo et al, Fong et al).  The chloro subfamily has a number of distinctive variations relative to other CAKs and PKL kinases, including the loss of a conserved salt bridge holding the N-terminal lobe together, and a divergent ATP binding motif (Kannan et al)


    Figure 1. Monomer of YP_614837.1, colored from blue (N-term) to red (C-term)

    The biological unit of YP_614837.1 is a dimer, as shown in Figure 2. Each monomer contributes two residues (Glu81 and His83) to a Zn binding site, which is located on the surface of the dimer interface.


    Figure 2. Dimer view of YP_614837.1, with the Zn binding site bridging the two symmetry-related monomers.

    Structural Overview

    CAK-chloro retains the basic CAK fold, previously defined by the structures of APH and ChoK (Scheeff and Bourne 2005; Kannan, Taylor et al. 2007).  There is a distinctive insertion in all CAK structures between helix E and the catalytic loop (PKA nomenclature is used for secondary structure elements in this discussion) consisting of two core helices, plus occasional additional secondary structure (Figure 3).  This structure essentially replaces the activation loop seen in the protein kinases, which is missing in CAKs.  This structure is present in CAK-chloro, and consists of the two core helices.  CAKs also have a distinctive c-terminal helical structure, and this is also present, and in a typical form, in CAK-chloro (Figure 3). CAK-chloro is structurally most similar to HSK2 (PDB:2ppq), with an RMSD of 3.3 angstroms across 263 aligned positions.


    Figure 3. Comparison of CAK-chloro, HSK2, and APH structures. The distinctive structures seen in CAKs are highlighted, with the insert between helix E and the catalytic loop in blue and the c-terminal structure in yellow.

    ATP Binding Pocket / Catalytic Residues

    The chloro subfamily has a number of distinctive ATP-binding pocket variations relative to other CAKs and PKL kinases.  These changes are suggestive of a somewhat altered mode of ATP binding relative to other CAK enzymes.

    The distinctive ion pair seen in most PKL kinases, which links strand 3 to helix C (Scheeff and Bourne 2005), is missing in the the CAK-chloro subfamily. This modification is also seen in the structurally similar HSK2 subfamily, but not in other members of the CAK family, where it is intact (usually as an R-E pair). The positively charged residue of the pair is important for interaction with the phosphate groups of bound ATP (it is replaced by methionine in CAK-chloro and theonine in HSK2) . It is plausible that R32 of strand 2 rescues the missing positive charge in CAK-chloro (Kannan, Taylor et al. 2007). A similar shift of the positive charge from strand 3 to strand 2 has been observed in the WNK protein kinase, with retention of ATP binding and catalytic activity (Min, Lee et al. 2004). However, it cannot be discerned that this is the case based on the available data, because R32, while highly conserved in sequence alignments (Kannan, Taylor et al. 2007), is not resolved in the structure (also, ATP is not present). The residues in the analogous position to R32 are resolved in both the HSK and APH structures, and in both cases their location and orientation are consistent with potential ATP interaction.  It is also possible that a positively charged residue is not strictly required for ATP interaction in all PKL kinases. HSK does not show a similar conservation of R at the equivalent position (Kannan, Taylor et al. 2007), and appears to lack clear compensation for the loss of positive charge in the ATP binding pocket. Also, at least one case is now known where a protein kinase with major ATP binding pocket alterations still retains activity (Mukherjee, Sharma et al. 2008). Remarkably, CAK-chloro and HSK both replace the glutamate of the ion pair with a conserved phenylalanine residue, though the side chain orientation differs between the two structures: in CAK-chloro, F61 faces towards the ATP-binding pocket; in HSK2 F65 is packed into the structure behind the DFG motif (DFY in HSK2).

    CAK-chloro also contains a distinctive change to an aromatic residue at Y34 (conserved in CAK-chloro as Y or F, Kannan et al.), a position that normally interacts with the adenine ring of ATP (V57 in PKA).  This change is not seen in any other PKL family, but it is likely that this residue forms a ring stacking interaction with the adenine ring in the ATP-bound form. Such ring stacking interactions with ATP are a common feature of the CAK family, though the specific residues involved differ in various structures.  Further, interaction of the adenine ring with Y34 might change the orientation of the ATP molecule such that the phosphate groups would be in a better position to interact with R32.  Thus, the movement of positive charge from strand 3 to strand 2 may have resulted in other adjustments in the ATP binding pocket.

    Other than the above changes, the active site of CAK-chloro is relatively typical for CAK family members.  The catalytic aspartate residue and metal binding residues are present and conserved, and the hydrogen bond network in the c-terminal subunit that supports the catalytic loop structure (Scheeff and Bourne 2005) is intact, as it is in most other CAKs.

     

    Ligand Summary



    References

    Reviews

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