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Structural Investigation of the Antibiotic and ATP-Binding Sites in Kanamycin Nucleotidyltransferase+Y$

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Structural Investigation of the Antibiotic and ATP-Binding Sites in Kanamycin Nucleotidyltransferase+Y$ Biochemistry 1995, 34, 13305-1331 1 13305 Structural Investigation of the Antibiotic and ATP-Binding Sites in Kanamycin Nucleotidyltransferase+y$ Lars C. Pedersen, Matthew M. Benning, and Hazel M. Holden* Institute for Enzyme Research, Graduate School, and Department of Biochemistry, Uniuersity of Wisconsin, Madison, Wisconsin 53705 Receiued June 28, 1995; Reuised Manuscript Receiued July 27, 1995@ ABSTRACT: Kanamycin nucleotidyltransferase (KNTase) is a plasmid-coded enzyme responsible for some types of bacterial resistance to aminoglycosides. The enzyme deactivates various antibiotics by transferring a nucleoside monophosphate group from ATP to the 4'-hydroxyl group of the drug. Detailed knowledge of the interactions between the protein and the substrates may lead to the design of aminoglycosides less susceptible to bacterial deactivation. Here we describe the structure of KNTase complexed with both the nonhydrolyzable nucleotide analog AMPCPP and kanamycin. Crystals employed in the investigation were grown from poly(etky1ene glycol) 2olutions and bFlonged to the space group P212121 with unit cell dimensions of a = 57.3 A, b = 102.2 ,A, c = 101.8 A, and one dimer in the asymmetric unit. Least- squares refinement of the model at 2.5 A resolution reduced the crystallographic R factor to 16.8%. The binding pockets for both the nucleotide and the antibiotic are extensively exposed to the solvent and are composed of amino acid residues contributed by both subunits in the dimer. There are few specific interactions between the protein and the adenine ring of the nucleotide; rather the AMPCPP molecule is locked into position by extensive hydrogen bonding between the a-, p-, and y-phosphates and protein side chains. This, in part, may explain the observation that the enzyme can utilize other nucleotides such as GTP and UTP. The 4'-hydroxyl group of the antibiotic is approximately 5 A from the a-phosphorus of the nucleotide and is in the proper orientation for a. single in-line displacement attack at the phosphorus. Glu 145, which lies within hydrogen-bonding distance of the 4'-hydroxyl group, is proposed to be the active site base. Aminoglycoside antibiotics consist of amino sugars linked to an aminocyclitol moiety through glycosidic bonds as no shown in Figure 1. The bactericidal effect of these com- pounds is believed to occur through their irreversible binding to the bacterial ribosomes (Moellering, 1983). Although there are newer, less toxic compounds on the market, the aminoglycosides still play useful roles in the treatment of serious infections caused by a broad range of microorganisms (Edson & Terrel, 1991; Johnson & Hardin, 1992). Unfor- tunately, due to past widespread usage of these antibiotics in clinical settings, bacterial strains have emerged that render these compounds ineffective. Resistance to aminoglycosides FIGURE1: General structure of the antibiotic kanamycin. Kana- is typically conferred by the enzymatic activity of plasmid- mycins A and B differ only at the 2'-position as indicated. Both coded acetyltransferases, phosphotransferases,and nucleoti- the 4'- and the 4"-hydroxyl groups are susceptible to nucleotid- dyltransferases [for a review, see Shaw et al. (1993)l. ylation by KNTase. Kanamycin nucleotidyltransferase (KNTase) is one such The three-dimensional structure of KNTase was recently enzyme and catalyzes the transfer of a nucleoside mono- determined by X-ray crystallographic techniques (Sakon et phosphate group from a nucleotide to the 4'-hydroxyl group al., 1993). From this structural study it was shown that the of kanamycin. First isolated from Staphylococcus aureus, enzyme was dimeric rather than monomeric as originally the enzyme can utilize ATP, GTP, or UTP as the nucleoside believed (Sadaie et al., 1980). The individual subunits of monophosphate donor and is capable of deactivating a KNTase are remarkably extended as shown in Figure 2a and number of different aminoglycosides (Le Goffic et al., can be described in terms of two structural domains. The N-terminal domain, delineated by Met 1 to Glu 127, is 1976a,b; Davies & Smith, 1978). characterized by a five-stranded mixed /3-pleated sheet whereas the C-terminal motif, formed by Ala 128 to Phe +This research was supported in part by a grant from the NIH 253, contains five a-helices, four of which form an up-and- (DK47814). down a-helical bundle. As can be seen in Figure 2b, the X-ray coordinates have been deposited with the Brookhaven Protein two subunits interact extensively by wrapping around one Data Bank under the filename 1KNY. * To whom correspondence should be addressed. another to form a rather large cavity which can easily @ Abstract published in Advance ACS Abstracts, October 1, 1995. accommodate a variety of aminoglycosides. On the basis 0006-2960/95/0434-13305$09.00/0 0 1995 American Chemical Society 13306 Biochemist?y, Vol. 34, No. 41, 1995 Pedersen et al. FIGURE2: Ribbon representation of the apo form of KNTase. Models for the individual monomer and the dimer of KNTase are shown in panels a (top) and b (bottom), respectively. The domains depicted in blue and yellow belong to subunit I; those in red and green represent subunit 11. This figure was prepared with the software package MOLSCRIPT (Kraulis, 1991). of this initial structural investigation, the two active sites These cells were stored at -70 "C prior to use. The enzyme for the KNTase homodimer were tentatively located and purification scheme employed was a modification of previ- shown to be formed by both subunits (Sakon et al., 1993). ously reported procedures and included an initial chromato- Here we describe the three-dimensional structure of graphic step with a DEAE-Sephadex-6505 column followed KNTase complexed with AMPCPP, a nonhydrolyzable by a TSK phenyl-SPW hydrophobic column and subsequent analog of ATP, and kanamycin. This structural investigation use of an HPLC Mono-Q HR10/10 column (Liao et al., 1986; provides a more complete description of the active site of Liao, 1991; Sadie et al., 1980; Sakon et al., 1993). The this enzyme and reveals both important protein-nucleotide purified protein was stored in 10 mM Tris-HC1, pH 7.5 at and protein-antibiotic interactions. Detailed structural -20 "C. Thus far it has not been possible to obtain X-ray information concerning the active site of KNTase may quality crystals for the wild-type enzyme. ultimately lead to the design of new and more effective aminoglycosides and possibly to the production of KNTase Prior to crystallization, the protein, at 15 mg/ml, was inhibitors. This is especially important in light of both the incubated with 10 mM kanamycin A for 1 h at 4 "C. X-ray alarming and rapid increase in the emergence of antibiotic- diffraction quality crystals were obtained by the hanging drop resistant bacterial strains and the lack of significant new method of vapor diffusion. For such experiments, 5 pL of antibiotics appearing on the pharmaceutical market. the protein- kanamycin solution was mixed on silanized coverslips with 5 pL of a solution containing 12.5% PEG MATERIALS AND METHODS 3350, 150 mM MgC12, 5 mM NaN3, and 100 mM Hepes Purijtcation and Ciystallization Procedures. The KNTase (pH 7.7). These droplets were equilibrated against the 12.5% mutant, D80Y, was expressed in the Escherichia coli PEG solution. Crystals generally appeared within 1 week overproducing strain BL2 1(DE3) carrying the expression with some achieving maximum dimensions of 0.3 mm x plasmid pX(T7)TKl (Studier & Moffatt, 1986; Liao, 1991). 0.3 mm x 0.7 mm. Active Site of Kanamycin Nucleotidyltransferase Biochemistry, Vol. 34, No. 41, 1995 13307 Table 1: Intensity Statistics for the Native X-ray Data resolution range (A) native overall 100.00-5.00 3.97 3.47 3.15 2.92 2.15 2.61 2.50 no. of measurements 51522 7655 7898 7331 6580 5992 5708 5299 5059 no. of independent reflections 20767 2513 2548 2590 2600 2611 2641 2626 2638 completeness of data (%) 81 84 89 90 88 83 16 IO 65 av intensity 457 1 9095 10000 5980 3266 1673 976 666 487 av u 438 366 499 473 443 424 42 1 429 442 R factor" (5%) 5 .O 3.5 3.4 4.5 6.8 11.1 17.0 21.9 29.1 Prior to X-ray data collection, the crystals were transferred Table 2: Least-Squares Refinement Statistics to a solution containing 14% PEG 3350, 150 mM MgC12, 5 resolution limits (A) 30.0-2.5 mM NaN3, 100 mM Hepes (pH 7.7), 20 mM kanamycin A, R factor" (%)a 16.8 and 5 mM adenosine 5'-a,P-methylenetriphosphate (AMP- no. of reflections used 19 405 no. of protein atoms 4187 CPP). To minimize crystal cracking, the concentration of no. of solvent atoms 46 AMPCPP was slowly increased to 10 mM and finally to 16 weighted rms deyiations from ideality mM over a period of 2 h. Crystals were soaked in this bond length (A) 0.014 kanamycin-AMPCPP solution for 24 h. On the basis of bond angle (deg) 2.5 precession photography, the crystals were shown to belong planarity (trigonal) (A) 0.006 planarity (other planes) (A) 0.012 to the space group P212121 with unit cell dimensions of a = torsional angleb (deg) 19.2 57.3 A, b = 102.2 A, c = 101.8 A, and one dimer in the R factor = ZIFo - FclE1F,J, where F, is the observed structure asymmetric unit. factor amplitude and F, is the calculated structure factor amplitude. X-ray Data Collection and Processing. X-ray diffraction The torsional angles were not restrained during the refinement. data were collected to 2.5 A resolution at 4 "C with a Siemens XlOOOD area detector system equipped with double- RESULTS AND DISCUSSION focusing mirrors. The X-ray source was Cu Ka radiation from a Rigaku RU200 rotating anode generator operated at The original model of KNTase was derived from crystals 50 kV and 50 mA.
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