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Aminoglycoside 2′′-Phosphotransferase IIIa (APH(2′′)-IIIa) Prefers GTP over ATP

STRUCTURAL TEMPLATES FOR NUCLEOTIDE RECOGNITION IN THE BACTERIAL AMINOGLYCOSIDE-2′′ KINASES*
  • Clyde A. Smith
    Correspondence
    To whom correspondence should be addressed. 2575 Sand Hill Rd., Menlo Park, CA 94025. Fax: 650-926-3292;
    Affiliations
    Stanford Synchrotron Radiation Lightsource, Stanford University, Menlo Park, California 94025
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  • Marta Toth
    Affiliations
    Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556
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  • Hilary Frase
    Affiliations
    Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556
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  • Laura J. Byrnes
    Footnotes
    Affiliations
    Stanford Synchrotron Radiation Lightsource, Stanford University, Menlo Park, California 94025
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  • Sergei B. Vakulenko
    Affiliations
    Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556
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  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grants AI057393 (to S. B. V.) and 5 P41 RR001209 (Center for Research Resources).
    This article contains supplemental Figs. S1–S5 and Tables S1–S3.
    2 Supported by the Science Undergraduate Laboratory Internship program. Present address: Dept. of Molecular Medicine, Cornell University, Ithaca, NY 14853.
Open AccessPublished:February 24, 2012DOI:https://doi.org/10.1074/jbc.M112.341206
      Contrary to the accepted dogma that ATP is the canonical phosphate donor in aminoglycoside kinases and protein kinases, it was recently demonstrated that all members of the bacterial aminoglycoside 2′′-phosphotransferase IIIa (APH(2′′)) aminoglycoside kinase family are unique in their ability to utilize GTP as a cofactor for antibiotic modification. Here we describe the structural determinants for GTP recognition in these enzymes. The crystal structure of the GTP-dependent APH(2′′)-IIIa shows that although this enzyme has templates for both ATP and GTP binding superimposed on a single nucleotide specificity motif, access to the ATP-binding template is blocked by a bulky tyrosine residue. Substitution of this tyrosine by a smaller amino acid opens access to the ATP template. Similar GTP binding templates are conserved in other bacterial aminoglycoside kinases, whereas in the structurally related eukaryotic protein kinases this template is less conserved. The aminoglycoside kinases are important antibiotic resistance enzymes in bacteria, whose wide dissemination severely limits available therapeutic options, and the GTP binding templates could be exploited as new, previously unexplored targets for inhibitors of these clinically important enzymes.

      Introduction

      Bacteria use kinase-like enzymes, known as aminoglycoside kinases or aminoglycoside phosphotransferases (APHs),
      The abbreviations used are: APH
      aminoglycoside phosphotransferase
      APH(2″)
      aminoglycoside-2″-phosphotransferase
      ePK
      eukaryotic protein kinase
      CK2
      casein kinase II
      CAMPK
      cyclic-AMP-dependent kinase
      NSM
      nucleotide selectivity motif
      ATPγS
      adenosine 5′-O-(thiotriphosphate)
      r.m.s.d.
      root mean square deviation
      AMPPNP
      adenosine 5′-(β,γ-imino)triphosphate
      AMPPCP
      adenosine 5'-(β,γ-methylene)triphosphate.
      for self-protection against clinically important aminoglycoside antibiotics. The aminoglycoside antibiotics, produced by soil microorganisms as part of an arsenal in the ongoing battle waged between soil bacteria, include the clinically relevant drugs gentamicin and kanamycin, (supplemental Fig. S1). The aminoglycosides are a family of broad-spectrum antibiotics targeting the bacterial ribosome and disrupting protein synthesis (
      • Moazed D.
      • Noller H.F.
      Interaction of antibiotics with functional sites in 16 S ribosomal RNA.
      ,
      • Karimi R.
      • Ehrenberg M.
      Dissociation rate of cognate peptidyl-tRNA from the A-site of hyper-accurate and error-prone ribosomes.
      ,
      • Wirmer J.
      • Westhof E.
      Molecular contacts between antibiotics and the 30 S ribosomal particle.
      ) and are used for the treatment of infections caused by both Gram-positive and Gram-negative bacteria (
      • Edson R.S.
      • Terrell C.L.
      The aminoglycosides.
      ). They all have a similar structure, consisting of a central aminocyclitol ring (the B ring) with aminoglycan groups (the A and C rings) attached to the B ring (supplemental Fig. S1). Several subfamilies of APH enzymes are known, named according to the position on the aminoglycoside at which they act, and their substrate/cofactor specificity (
      • Wright G.D.
      • Thompson P.R.
      Aminoglycoside phosphotransferases: proteins, structure and mechanism.
      ,
      • Wright G.D.
      Aminoglycoside-modifying enzymes.
      ,
      • Smith C.A.
      • Baker E.N.
      Aminoglycoside antibiotic resistance by enzymatic deactivation.
      ,
      • Vakulenko S.B.
      • Mobashery S.
      Versatility of aminoglycosides and prospects for their future.
      ,
      • Ramirez M.S.
      • Tolmasky M.E.
      Aminoglycoside modifying enzymes.
      ), with the APH(3′) and APH(2″) families being the most prevalent. Enzymes in these two families have the capability of conferring resistance to a wide range of aminoglycosides, yet no two enzymes have quite the same substrate specificity profile, raising important questions with regard to what structural features are important for this selectivity. The APH enzymes phosphorylate free hydroxyl groups on the antibiotics, which interferes with the subsequent binding of these molecules to their ribosomal target. The phosphate group is typically derived from ATP and the phosphoryl transfer mechanism involves a conserved aspartate residue that initially orients the substrate hydroxyl group (
      • Kim C.
      • Mobashery S.
      Phosphoryl transfer by aminoglycoside 3′-phosphotransferases and manifestation of antibiotic resistance.
      ) and subsequently acts as a proton acceptor once cleavage of the phosphorus-oxygen bond has begun (
      • Valiev M.
      • Kawai R.
      • Adams J.A.
      • Weare J.H.
      The role of the putative catalytic base in the phosphoryl transfer reaction in a protein kinase. First-principles calculations.
      ). The triphosphate moiety of the nucleotide is stabilized by the presence of two magnesium ions that bridge the nucleotide and the enzyme and neutralize some of the negative charge on the triphosphate.
      Structurally, the APHs are similar to the eukaryotic protein kinase (ePK) catalytic domain, and although there is limited overall sequence identity, the APHs contain a number of kinase-specific sequence motifs related to nucleotide binding and phosphoryl transfer. It is generally accepted that the highly regulated ePKs evolved from a simpler non-regulated ancestral kinase that may have resembled the APHs (
      • Taylor S.S.
      • Kornev A.P.
      Protein kinases. Evolution of dynamic regulatory proteins.
      ). Bacterial and archaeal genomes also contain a wide range of protein kinases (bacterial protein kinases (bPKs) and archaeal protein kinases (aPKs)) that share sequence and structural similarities with the ePKs (
      • Leonard C.J.
      • Aravind L.
      • Koonin E.V.
      Novel families of putative protein kinases in bacteria and archaea. Evolution of the “eukaryotic” protein kinase superfamily.
      ,
      • Pereira S.F.
      • Goss L.
      • Dworkin J.
      Eukaryote-like serine/threonine kinases and phosphatases in bacteria.
      ,
      • Ortiz-Lombardía M.
      • Pompeo F.
      • Boitel B.
      • Alzari P.M.
      Crystal structure of the catalytic domain of the PknB serine/threonine kinase from Mycobacterium tuberculosis.
      ,
      • LaRonde-LeBlanc N.
      • Wlodawer A.
      Crystal structure of A. fulgidus Rio2 defines a new family of serine protein kinases.
      ,
      • Laronde-Leblanc N.
      • Guszczynski T.
      • Copeland T.
      • Wlodawer A.
      Structure and activity of the atypical serine kinase Rio1.
      ,
      • Zheng J.
      • He C.
      • Singh V.K.
      • Martin N.L.
      • Jia Z.
      Crystal structure of a novel prokaryotic Ser/Thr kinase and its implication in the Cpx stress response pathway.
      ), and phosphorylation by protein kinases is now seen as a universal process in all kingdoms of life (
      • Cozzone A.J.
      ATP-dependent protein kinases in bacteria.
      ).
      The protein kinases (PKs; collectively the ePKs, bPKs, and aPKs) almost always use ATP as a cofactor for phosphoryl transfer (
      • Shugar D.
      The NTP phosphate donor in kinase reactions. Is ATP a monopolist?.
      ) with few exceptions, notably casein kinase II (CK2) (
      • Niefind K.
      • Pütter M.
      • Guerra B.
      • Issinger O.G.
      • Schomburg D.
      GTP plus water mimic ATP in the active site of protein kinase CK2.
      ), calcium/calmodulin-dependent protein kinase II (
      • Bostrom S.L.
      • Dore J.
      • Griffith L.C.
      CaMKII uses GTP as a phosphate donor for both substrate and autophosphorylation.
      ), mst-3 kinase (
      • Schinkmann K.
      • Blenis J.
      Cloning and characterization of a human STE20-like protein kinase with unusual cofactor requirements.
      ), and protein kinase Cδ, which shows an increase in autophosphorylation with GTP (
      • Gschwendt M.
      • Kittstein W.
      • Kielbassa K.
      • Marks F.
      Protein kinase Cδ accepts GTP for autophosphorylation.
      ). Our recent work on the APHs from the aminoglycoside-2″-phosphotransferase [APH(2″)] subfamily (
      • Toth M.
      • Chow J.W.
      • Mobashery S.
      • Vakulenko S.B.
      Source of phosphate in the enzymic reaction as a point of distinction among aminoglycoside 2″-phosphotransferases.
      ) have shown that not only are these enzymes capable of using GTP as the phosphate source but that two members of this family (APH(2″)-Ia and APH(2″)-IIIa) utilize GTP exclusively in vivo. The ability of the APH(2″) enzymes to use GTP as a cofactor is unexpected, as APHs were also considered to be exclusively ATP-dependent (
      • Shugar D.
      The NTP phosphate donor in kinase reactions. Is ATP a monopolist?.
      ,
      • Shakya T.
      • Wright G.D.
      Nucleotide selectivity of antibiotic kinases.
      ). This raises a number of questions related to nucleotide binding and catalysis in the aminoglycoside kinases and in the protein kinases in general. What are the structural determinants of nucleotide selectivity in these enzymes, and why is it that the eukaryotic kinases have evolved to be almost exclusively ATP-dependent, whereas some bacterial aminoglycoside kinases can be exclusively GTP-dependent, exclusively ATP-dependent, or possess the ability to utilize both of these substrates? In an attempt to answer some of these questions we have solved the crystal structure of APH(2″)-IIIa, an aminoglycoside kinase that uses only GTP as the phosphate source. We provide evidence for the existence of a structural template for GTP recognition in all bacterial aminoglycoside kinases from the APH(2″) subfamily, a template that is also conserved in other APH enzymes yet is poorly conserved in the majority of bacterial Ser/Thr kinases and eukaryotic protein kinases.

      DISCUSSION

      The bacterial aminoglycoside kinases (APHs) include several families of clinically important enzymes that produce resistance to aminoglycoside antibiotics by the transfer of phosphate groups to free hydroxyl groups on the drugs. Structurally, the APHs are similar to the ePKs, enzymes that catalyze protein phosphorylation leading to the regulation of multiple cellular processes. Until recently, it was widely accepted that ATP is the source of phosphate for these enzymes in vivo, and structural elements involved in ATP binding were extensively studied in ePKs and to a lesser degree in bacterial APHs. For APH(3′)-IIIa it was established that in conjunction with hydrogen bonding to the hinge peptide and π-π stacking interactions with aromatic residues, a methionine residue in the gatekeeper position played a key role in adenine recognition in this enzyme. Significantly more information is available on the structural determinants of ATP binding in the ePKs. Comparative analysis of the ePKs has demonstrated that in addition to the adenine recognition site on the hinge peptide, which has long been used as a template for the development of kinase inhibitors (
      • Adams J.
      • Huang P.
      • Patrick D.
      A strategy for the design of multiplex inhibitors for kinase-mediated signaling in angiogenesis.
      ,
      • Noble M.E.
      • Endicott J.A.
      • Johnson L.N.
      Protein kinase inhibitors. Insights into drug design from structure.
      ,
      • Traxler P.
      • Furet P.
      Strategies toward the design of novel and selective protein tyrosine kinase inhibitors.
      ,
      • García-Echeverría C.
      • Traxler P.
      • Evans D.B.
      ATP site-directed competitive and irreversible inhibitors of protein kinases.
      ), these enzymes contain a number of very highly conserved sequence motifs related to the binding and orientation of the triphosphate of ATP and to phosphoryl transfer (
      • Boehr D.D.
      • Thompson P.R.
      • Wright G.D.
      Molecular mechanism of aminoglycoside antibiotic kinase APH(3′)-IIIa. Roles of conserved active site residues.
      ). These motifs include the glycine-rich G-loop, the catalytic loop, and the activation segment. The G-loop is critical for activity and correctly positions the γ-phosphate of ATP for attack on the hydroxyl acceptor (
      • Aimes R.T.
      • Hemmer W.
      • Taylor S.S.
      Serine-53 at the tip of the glycine-rich loop of cAMP-dependent protein kinase. Role in catalysis, P-site specificity, and interaction with inhibitors.
      ,
      • Barouch-Bentov R.
      • Che J.
      • Lee C.C.
      • Yang Y.
      • Herman A.
      • Jia Y.
      • Velentza A.
      • Watson J.
      • Sternberg L.
      • Kim S.
      • Ziaee N.
      • Miller A.
      • Jackson C.
      • Fujimoto M.
      • Young M.
      • Batalov S.
      • Liu Y.
      • Warmuth M.
      • Wiltshire T.
      • Cooke M.P.
      • Sauer K.
      A conserved salt bridge in the G loop of multiple protein kinases is important for catalysis and for in vivo Lyn function.
      ) by firmly anchoring the α- and β-phosphates. The catalytic loop (HRDXXXXN) contains the essential aspartate residue (the underline in the consensus sequence), which acts as the catalytic base and ultimately accepts the proton from the hydroxyl substrate. The activation segment (XDFGXYXAPEX) is a loop of variable length and sequence, where the DFG and APE motifs represent anchor points at each end of the flexible loop. This loop undergoes a large scale conformational change generally in response to phosphorylation (
      • Taylor S.S.
      • Kornev A.P.
      Protein kinases. Evolution of dynamic regulatory proteins.
      ,
      • Johnson L.N.
      Protein kinase inhibitors. Contributions from structure to clinical compounds.
      ,
      • Boehr D.D.
      • Farley A.R.
      • LaRonde F.J.
      • Murdock T.R.
      • Wright G.D.
      • Cox J.R.
      Establishing the principles of recognition in the adenine binding region of an aminoglycoside antibiotic kinase [APH(3′)-IIIa].
      ).
      These three sequence motifs are also conserved in the APHs (supplemental Fig. S4). First, all of the aminoglycoside kinases have a G-loop equivalent to the PK G-loop. There is some variation in the G-loop sequence between the APHs and the PKs, but structurally the loop plays the same role, folding over the triphosphate, positioning the γ-phosphate correctly, and promoting phosphoryl transfer (
      • Thompson P.R.
      • Boehr D.D.
      • Berghuis A.M.
      • Wright G.D.
      Mechanism of aminoglycoside antibiotic kinase APH(3′)-IIIa. Role of the nucleotide positioning loop.
      ). The catalytic aspartate residue in the DXXXXN motif is also spatially conserved (Asp-196 in APH(2″)-IIIa) and is essential for activity in the APH enzymes (
      • Hon W.C.
      • McKay G.A.
      • Thompson P.R.
      • Sweet R.M.
      • Yang D.S.
      • Wright G.D.
      • Berghuis A.M.
      Structure of an enzyme required for aminoglycoside antibiotic resistance reveals homology to eukaryotic protein kinases.
      ,
      • Kocabiyik S.
      • Perlin M.H.
      Site-specific mutations of conserved C-terminal residues in aminoglycoside 3′-phosphotransferase II. Phenotypic and structural analysis of mutant enzymes.
      ). Once again the sequences show some variation particularly at the C-terminal end where some of the APHs (including APH(2″)-IIIa) have a histidine residue (His-201) instead of an asparagine, which coordinates to one of the magnesium ions (Fig. 3, A and B). The activation segment is partially conserved, with the DFG motif present in all the APH enzymes, and the aspartate residue (Asp-218) involved in magnesium binding (Fig. 3B). However, the aminoglycoside kinases do not possess the long PK-like activation segment and are not subject to regulation by phosphorylation (
      • Taylor S.S.
      • Kornev A.P.
      Protein kinases. Evolution of dynamic regulatory proteins.
      ). Instead, the APHs have an open cleft in roughly the same location that is the site of aminoglycoside substrate binding (
      • Young P.G.
      • Walanj R.
      • Lakshmi V.
      • Byrnes L.J.
      • Metcalf P.
      • Baker E.N.
      • Vakulenko S.B.
      • Smith C.A.
      The crystal structures of substrate and nucleotide complexes of Enterococcus faecium aminoglycoside-2″-phosphotransferase-IIa [APH(2″)-IIa] provide insights into substrate selectivity in the APH(2″) subfamily.
      ,
      • Shi K.
      • Houston D.R.
      • Berghuis A.M.
      Crystal structures of antibiotic-bound complexes of aminoglycoside 2″-phosphotransferase IVa highlight the diversity in substrate binding modes among aminoglycoside kinases.
      ,
      • Fong D.H.
      • Berghuis A.M.
      Substrate promiscuity of an aminoglycoside antibiotic resistance enzyme via target mimicry.
      ). The structures of the nucleotide-bound complexes of six APH enzymes are known, five with ATP or an analog bound, APH(2″)-IIa (
      • Young P.G.
      • Walanj R.
      • Lakshmi V.
      • Byrnes L.J.
      • Metcalf P.
      • Baker E.N.
      • Vakulenko S.B.
      • Smith C.A.
      The crystal structures of substrate and nucleotide complexes of Enterococcus faecium aminoglycoside-2″-phosphotransferase-IIa [APH(2″)-IIa] provide insights into substrate selectivity in the APH(2″) subfamily.
      ), APH(3′)-Ia (PDB code 3R78), APH(3″)-IIIa (
      • Burk D.L.
      • Hon W.C.
      • Leung A.K.
      • Berghuis A.M.
      Structural analyses of nucleotide binding to an aminoglycoside phosphotransferase.
      ), APH(9)-Ia (
      • Fong D.H.
      • Lemke C.T.
      • Hwang J.
      • Xiong B.
      • Berghuis A.M.
      Structure of the antibiotic resistance factor spectinomycin phosphotransferase from Legionella pneumophila.
      ), and Rv3168 (
      • Kim S.
      • Nguyen C.M.
      • Kim E.J.
      • Kim K.J.
      Crystal structure of Mycobacterium tuberculosis Rv3168. A putative aminoglycoside antibiotics resistance enzyme.
      ), and now the GDP complex of APH(2″)-IIIa, and in all cases, irrespective of the identity of the nucleotide (adenine or guanine), the triphosphate moiety is bound in the same location and by a highly conserved set of structural determinants.
      Our recent kinetic studies demonstrated that unlike the majority of the ePKs, all four members of bacterial aminoglycoside-2″ phosphotransferase subfamily are capable of utilizing GTP as the phosphate donor (
      • Toth M.
      • Chow J.W.
      • Mobashery S.
      • Vakulenko S.B.
      Source of phosphate in the enzymic reaction as a point of distinction among aminoglycoside 2″-phosphotransferases.
      ). Two of these enzymes are able to use both nucleotides, with APH(2″)-IIa having a slight preference for ATP over GTP, whereas the other, APH(2″)-IVa, has similar relative affinities for these NTPs (Table 3). The two other members of the family, APH(2″)-Ia and APH(2″)-IIIa, are unique in their ability to use GTP exclusively, their respective relative affinities for GTP 250- and 400-fold greater than that for ATP (Table 3). Moreover, the relative affinity of APH(2″)-Ia and APH(2″)-IIIa for GTP is significantly higher than the relative affinity for ATP of various ATP-specific APHs and ePKs. The crystal structure of the Mg2GDP complex of APH(2″)-IIIa represents the first structure of one of these GTP-specific aminoglycoside kinases and offers insights into how this nucleotide can be accommodated in a binding site thought to be fine-tuned to recognize only ATP. By superimposing recognition templates for both adenine and guanine on hinge peptide, this family of enzymes has gained the ability to recognize both ATP and GTP to the point where the APH(2″)-Ia and APH(2″)-IIIa aminoglycoside kinases have evolved to exclusively bind GTP and use it as the source of phosphate in phosphoryl transfer reactions. Comparative analyses of the known APH(2″) enzyme structures shows that the guanine specificity template is identical in all three enzymes. Although the guanine-specific template is positioned slightly farther toward the outside edge of the nucleotide binding cleft relative to the ATP-specific template, the conformation of the hinge allows for the productive binding of either GTP or ATP, such that triphosphate binding is essentially identical irrespective of the identity of the nucleotide. Moreover, our analyses also show that a guanine specificity template also exists on the hinge region of enzymes from other APH families, although these enzymes have not been tested for the ability to utilize GTP, with the one exception. It has been reported recently that APH(4)-Ia has the ability to use both ATP and GTP (
      • Stogios P.J.
      • Shakya T.
      • Evdokimova E.
      • Savchenko A.
      • Wright G.D.
      Structure and function of APH(4)-Ia, a hygromycin B resistance enzyme.
      ), and it is highly likely that the same template involved the L3 residue and the L3-L5 water binding pocket will be used to accommodate the guanine ring, although confirmation of this must await further structural information.
      Analysis of the NSM of all PKs, which have nucleotide bound and whose structures are available in the Protein Date Bank, demonstrates that, with the exception of CK2, which has a similar NSM and a conserved guanine-specificity template comparable with the APHs, all other PKs show a marked difference in their NSMs. Perhaps the most important difference between the APHs and the PKs is in the relative position of the binding sites for the nucleotide base and the triphosphate (the G-loop). When APH(2″)-IIIa and CAMPK are superimposed based on strands β1 and β2 and the G-loop, the CAMPK-like NSM is substantially translated toward the core subdomain, placing its adenine-specific template ∼3 Å from the APH adenine-specific template and almost directly on top of the APH(2″)-IIIa guanine specificity template. The L3 residue is an additional 3 Å farther away (Fig. 5B). For guanine to make viable hydrogen-bonding interactions with the L3 residue in a CAMPK-like NSM, the triphosphate moiety of the GTP would be forced to move away from the G-loop, thus giving rise to an potentially unproductive complex. Because it may not be possible to satisfy the hydrogen bonding requirements of both specificity elements (the G-loop and the guanine-specific template) simultaneously, the affinity for GTP might be expected to be low in the majority of the PKs. Although individually the two nucleotide binding elements (the G-loop and the L1-L3 portion of the NSM) are similar in conformation in the PKs and the APHs, it is the spatial disposition of these two elements that is critical in determining which nucleotide is able to bind in an active configuration. Not only must the nucleotide base be anchored firmly to the NSM, but the triphosphate group must interact with the G-loop in such a way that the γ-phosphate is presented to the incoming substrate. This is equally true in the PKs and the APHs. The PKs have clearly evolved a nucleotide binding site specifically tuned to accept ATP in a productive manner, and given the structural repositioning of the NSM in these enzymes, it seems unlikely that GTP would be a viable substrate for most PKs. This clearly seems to be the case given the preponderance of evidence for ATP being the canonical phosphate donor in these enzymes and the comparative rarity of GTP utilization by kinases.
      Detailed analysis of the available structures of APHs along with bacterial and eukaryotic protein kinases indicates that templates for the binding of GTP are both structurally and spatially conserved within the aminoglycoside phosphotransferase superfamily but not in the PKs. At present there are no clinically available inhibitors of APHs aimed at either the ATP or the aminoglycoside substrate binding sites. Because the APH enzymes seem to be quite promiscuous with respect to their aminoglycoside substrate profiles (
      • Shi K.
      • Houston D.R.
      • Berghuis A.M.
      Crystal structures of antibiotic-bound complexes of aminoglycoside 2″-phosphotransferase IVa highlight the diversity in substrate binding modes among aminoglycoside kinases.
      ), the design of a general inhibitor that could act against a large number of the APHs seems impracticable. In addition, the prospects for designing inhibitors targeted at the ATP site are also poor due to the structural similarities between the ATP binding templates of APHs and ePKs, as such inhibitors would be expected to be highly toxic as they could potentially inhibit a wide array of human protein kinases. The discovery that there exist consecutive templates for selective adenine and guanine binding within the hinge region of the APH enzymes, which show a much greater variability in ePKs, provides a rationale for the design of selective inhibitors of the APHs. Because the structural templates for GTP and ATP binding by aminoglycoside kinases overlap to some extent, small molecules targeting the GTP specificity template of APHs are also expected to block access to the ATP binding template of these enzymes, ensuring efficient inhibition. Moreover, inhibitors targeting GTP binding in the APHs are expected to exhibit low toxicity as they would not bind promiscuously to the majority of human ePKs and affect their activity. Furthermore, because the mode of guanine binding in the APHs differs markedly from other human GTP-dependent enzymes including the small GTPases and the G proteins, cross-reactivity with these enzymes would also be minimal. Such inhibitors could then be co-administered with existing aminoglycosides, and this would be a major breakthrough in the treatment of life-threatening infections caused by multidrug-resistant microorganisms.

      Acknowledgments

      Portions of this research were carried out at the Stanford Synchrotron Radiation Lightsource, a national user facility operated by Stanford University on behalf of the United States Department of Energy, Office of Basic Energy Sciences. The Stanford Synchrotron Radiation Lightsource Structural Molecular Biology Program is supported by the Department of Energy (Offices of Basic Energy Sciences and Biological and Environmental Research) and by the National Institutes of Health (Center for Research Resources, National Institute of General Medical Sciences).

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