1pi4: Difference between revisions
New page: left|200px<br /><applet load="1pi4" size="450" color="white" frame="true" align="right" spinBox="true" caption="1pi4, resolution 1.39Å" /> '''Structure of N289A m... |
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[[Image:1pi4.gif|left|200px]]<br /><applet load="1pi4" size=" | [[Image:1pi4.gif|left|200px]]<br /><applet load="1pi4" size="350" color="white" frame="true" align="right" spinBox="true" | ||
caption="1pi4, resolution 1.39Å" /> | caption="1pi4, resolution 1.39Å" /> | ||
'''Structure of N289A mutant of AmpC in complex with SM3, a phenylglyclboronic acid bearing the cephalothin R1 side chain'''<br /> | '''Structure of N289A mutant of AmpC in complex with SM3, a phenylglyclboronic acid bearing the cephalothin R1 side chain'''<br /> | ||
==Overview== | ==Overview== | ||
Beta-lactamases are the most widespread resistance mechanism to | Beta-lactamases are the most widespread resistance mechanism to beta-lactam antibiotics, such as the penicillins and cephalosporins. Transition-state analogues that bind to the enzymes with nanomolar affinities have been introduced in an effort to reverse the resistance conferred by these enzymes. To understand the origins of this affinity, and to guide design of future inhibitors, double-mutant thermodynamic cycle experiments were undertaken. An unexpected hydrogen bond between the nonconserved Asn289 and a key inhibitor carboxylate was observed in the X-ray crystal structure of a 1 nM inhibitor (compound 1) in complex with AmpC beta-lactamase. To investigate the energy of this hydrogen bond, the mutant enzyme N289A was made, as was an analogue of 1 that lacked the carboxylate (compound 2). The differential affinity of the four different protein and analogue complexes indicates that the carboxylate-amide hydrogen bond contributes 1.7 kcal/mol to overall binding affinity. Synthesis of an analogue of 1 where the carboxylate was replaced with an aldehyde led to an inhibitor that lost all this hydrogen bond energy, consistent with the importance of the ionic nature of this hydrogen bond. To investigate the structural bases of these energies, X-ray crystal structures of N289A/1 and N289A/2 were determined to 1.49 and 1.39 A, respectively. These structures suggest that no significant rearrangement occurs in the mutant versus the wild-type complexes with both compounds. The mutant enzymes L119A and L293A were made to investigate the interaction between a phenyl ring in 1 and these residues. Whereas deletion of the phenyl itself diminishes affinity by 5-fold, the double-mutant cycles suggest that this energy does not come through interaction with the leucines, despite the close contact in the structure. The energies of these interactions provide key information for the design of improved inhibitors against beta-lactamases. The high magnitude of the ion-dipole interaction between Asn289 and the carboxylate of 1 is consistent with the idea that ionic interactions can provide significant net affinity in inhibitor complexes. | ||
==About this Structure== | ==About this Structure== | ||
1PI4 is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli] with PO4, K and SM3 as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Beta-lactamase Beta-lactamase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.5.2.6 3.5.2.6] Full crystallographic information is available from [http:// | 1PI4 is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli] with <scene name='pdbligand=PO4:'>PO4</scene>, <scene name='pdbligand=K:'>K</scene> and <scene name='pdbligand=SM3:'>SM3</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Beta-lactamase Beta-lactamase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.5.2.6 3.5.2.6] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1PI4 OCA]. | ||
==Reference== | ==Reference== | ||
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[[Category: Escherichia coli]] | [[Category: Escherichia coli]] | ||
[[Category: Single protein]] | [[Category: Single protein]] | ||
[[Category: Focia, P | [[Category: Focia, P J.]] | ||
[[Category: Minasov, G.]] | [[Category: Minasov, G.]] | ||
[[Category: Roth, T | [[Category: Roth, T A.]] | ||
[[Category: Shoichet, B | [[Category: Shoichet, B K.]] | ||
[[Category: K]] | [[Category: K]] | ||
[[Category: PO4]] | [[Category: PO4]] | ||
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[[Category: enzyme inhibitor complex]] | [[Category: enzyme inhibitor complex]] | ||
''Page seeded by [http:// | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 14:29:00 2008'' | ||
Revision as of 15:29, 21 February 2008
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Structure of N289A mutant of AmpC in complex with SM3, a phenylglyclboronic acid bearing the cephalothin R1 side chain
OverviewOverview
Beta-lactamases are the most widespread resistance mechanism to beta-lactam antibiotics, such as the penicillins and cephalosporins. Transition-state analogues that bind to the enzymes with nanomolar affinities have been introduced in an effort to reverse the resistance conferred by these enzymes. To understand the origins of this affinity, and to guide design of future inhibitors, double-mutant thermodynamic cycle experiments were undertaken. An unexpected hydrogen bond between the nonconserved Asn289 and a key inhibitor carboxylate was observed in the X-ray crystal structure of a 1 nM inhibitor (compound 1) in complex with AmpC beta-lactamase. To investigate the energy of this hydrogen bond, the mutant enzyme N289A was made, as was an analogue of 1 that lacked the carboxylate (compound 2). The differential affinity of the four different protein and analogue complexes indicates that the carboxylate-amide hydrogen bond contributes 1.7 kcal/mol to overall binding affinity. Synthesis of an analogue of 1 where the carboxylate was replaced with an aldehyde led to an inhibitor that lost all this hydrogen bond energy, consistent with the importance of the ionic nature of this hydrogen bond. To investigate the structural bases of these energies, X-ray crystal structures of N289A/1 and N289A/2 were determined to 1.49 and 1.39 A, respectively. These structures suggest that no significant rearrangement occurs in the mutant versus the wild-type complexes with both compounds. The mutant enzymes L119A and L293A were made to investigate the interaction between a phenyl ring in 1 and these residues. Whereas deletion of the phenyl itself diminishes affinity by 5-fold, the double-mutant cycles suggest that this energy does not come through interaction with the leucines, despite the close contact in the structure. The energies of these interactions provide key information for the design of improved inhibitors against beta-lactamases. The high magnitude of the ion-dipole interaction between Asn289 and the carboxylate of 1 is consistent with the idea that ionic interactions can provide significant net affinity in inhibitor complexes.
About this StructureAbout this Structure
1PI4 is a Single protein structure of sequence from Escherichia coli with , and as ligands. Active as Beta-lactamase, with EC number 3.5.2.6 Full crystallographic information is available from OCA.
ReferenceReference
Thermodynamic cycle analysis and inhibitor design against beta-lactamase., Roth TA, Minasov G, Morandi S, Prati F, Shoichet BK, Biochemistry. 2003 Dec 16;42(49):14483-91. PMID:14661960
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