Crystallization of soluble proteins in vapor diffusion for x-ray crystallography

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193

274

dAlthough ␤-lactams have been the most effective class of antibacterial agents used in clinical practice for the past half century, their effectiveness on Gram-negative bacteria has been eroded due to the emergence and spread of ␤-lactamase enzymes that are not affected by currently marketed ␤-lactam/␤-lactamase inhibitor combinations. Avibactam is a novel, covalent, non-␤-lactam ␤-lactamase inhibitor presently in clinical development in combination with either ceftaroline or ceftazidime. In vitro studies show that avibactam may restore the broad-spectrum activity of cephalosporins against class A, class C, and some class D ␤-lactamases. Here we describe the structures of two clinically important ␤-lactamase enzymes bound to avibactam, the class A CTX-M-15 extended-spectrum ␤-lactamase and the class C Pseudomonas aeruginosa AmpC ␤-lactamase, which together provide insight into the binding modes for the respective enzyme classes. The structures reveal similar binding modes in both enzymes and thus provide a rationale for the broad-spectrum inhibitory activity of avibactam. Identification of the key residues surrounding the binding pocket allows for a better understanding of the potency of this scaffold. Finally, avibactam has recently been shown to be a reversible inhibitor, and the structures provide insights into the mechanism of avibactam recyclization. Analysis of the ultra-high-resolution CTX-M-15 structure suggests how the deacylation mechanism favors recyclization over hydrolysis.

Section: Methodsmentioning

confidence: 99%

Structural Insight into Potent Broad-Spectrum Inhibition with Reversible Recyclization Mechanism: Avibactam in Complex with CTX-M-15 and Pseudomonas aeruginosa AmpC β-Lactamases

Lahiri

Mangani

Durand-Réville

et al. 2013

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193

274

“…Preliminary crystallization trials were performed with a 10-mg/ml protein solution in 100 mM Tris-H 2 SO 4 buffer at pH 7.0 using commercial screens (Crystal Screen 1 and 2; Hampton Research) and provided spherulites and very small and ill-formed crystals with two different precipitant solutions, 0.8 M Na,K L-tartrate tetrahydrate in 0.1 M HEPES, pH 7.5 (solution 1), and 2.0 M (NH 4 ) 2 SO 4 plus 2% (vol/vol) polyethylene glycol 400 (PEG 400) in 0.1 M HEPES, pH 7.5 (solution 2) (in all cases, 2-l drops of the protein solution were mixed with an equal amount of the precipitant solution). Diffraction-quality crystals were obtained by repeated cycles of micro-and macroseeding in a sitting drop setup (2) by using the crystals obtained from solution 1 seeded in a drop made by 1 l of a 10-mg/ml OXA-46 solution in ultrapure water and 1 l of a 1 M Na,K L-tartrate tetrahydrate solution in 50 mM HEPES at pH 7.5 and 2 to 4% (vol/vol) PEG 400 as precipitants. The crystallization plates were stored at 20°C.…”

Section: Methodsmentioning

confidence: 99%

Crystal Structure of the Narrow-Spectrum OXA-46 Class D β-Lactamase: Relationship between Active-Site Lysine Carbamylation and Inhibition by Polycarboxylates

Docquier

Benvenuti

Calderone

et al. 2010

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Class D ␤-lactamases represent a heterogeneous group of active-site serine ␤-lactamases that show an extraordinary panel of functional features and substrate profiles, thus representing relevant models for biochemical and structural studies. OXA-46 is a narrow-spectrum enzyme belonging to the OXA-2 subgroup which was found in a Pseudomonas aeruginosa clinical isolate from northern Italy. In this work, we obtained the three-dimensional structure of OXA-46, which shows the overall fold of active serine ␤-lactamases and a dimeric quaternary structure. Significant differences with currently available structures of class D ␤-lactamases were found in the loops located close to the active site, which differ in length and conformation. Interestingly, the three subunits present in the asymmetric unit showed some structural heterogeneity, only one of which presented a carbamylated lysine recognized as an important functional feature of class D enzymes. The carbamylation state of residue Lys75 appeared to be associated with different shapes and dimensions of the active site. Moreover, a tartrate molecule from the crystallization buffer was found in the active site of the noncarbamylated subunits, which interacts with catalytically relevant residues. The OXA-46 crystal asymmetric units thus interestingly present the structures of the free carbamylated active site and of the ligand-bound uncarbamylated active site, offering the structural basis for investigating the potential of new scaffolds of ␤-lactamase inhibitors.Class D ␤-lactamases are a group of bacterial enzymes involved in resistance to ␤-lactam antibiotics that show an extraordinary structural and functional diversity. Indeed, these enzymes share only nine strictly conserved residues and may behave as narrow-spectrum oxacillinases (e.g., OXA-1, OXA-2, and FUS-1 [OXA-85]) or exhibit an extended spectrum of activity that includes oxyimino-cephalosporins (e.g., OXA-32 and OXA-11, which are OXA-2-and OXA-10-derivatives, respectively, and OXA-18, an OXA-type enzyme with intrinsic extended-spectrum ␤-lactamase [ESBL] properties) or even hydrolyze the most recent carbapenem antibiotics (e.g., OXA-23, OXA-24, OXA-48, and OXA-58) (20, 24). The emergence of class D ␤-lactamases that show extended-spectrum or carbapenemase activity has contributed to the increased clinical relevance of these resistance determinants (15,20,24,26).OXA-46 was identified in a multidrug-resistant Pseudomonas aeruginosa clinical isolate from Pavia (northern Italy), which also produced a metallo-␤-lactamase, and is encoded by an integron-borne gene cassette (9). On the basis of sequence similarity, OXA-46 belongs to the OXA-2 subgroup and shares 77.8% protein sequence identity with the latter, showed a narrow-spectrum substrate profile, and is inhibited by carbapenems, tazobactam and, to lesser extent, clavulanate (9). Notably, OXA-46 is poorly inhibited by NaCl, in comparison with other OXA-type enzymes, suggesting structural differences in the active-site region, as chloride ions have been hypoth...

“…BJP-1 was concentrated to 10 mg/ml, and the purification buffer was changed to 0.1 M Tris-HCl (pH 8.5) using a Microcon 10-kDa-cutoff ultrafiltration device (Millipore, Bedford, MA). The crystallization trials were performed using the sittingdrop method (96-well CrystalEX plates; Corning) (6). The drops consisted of 2 l protein solution and 2 l reservoir solution equilibrated at room temperature (20°C) against a reservoir volume of 100 l. The initial screens tested were Crystal Screen, Crystal Screen 2, and Grid Screen Ammonium Sulfate (Hampton Research, Aliso Viejo, CA).…”

Section: Methodsmentioning

confidence: 99%

High-Resolution Crystal Structure of the Subclass B3 Metallo-β-Lactamase BJP-1: Rational Basis for Substrate Specificity and Interaction with Sulfonamides

Docquier

Benvenuti

Calderone

et al. 2010

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Metallo-␤-lactamases (MBLs) are important enzymatic factors in resistance to ␤-lactam antibiotics that show important structural and functional heterogeneity. BJP-1 is a subclass B3 MBL determinant produced by Bradyrhizobium japonicum that exhibits interesting properties. BJP-1, like CAU-1 of Caulobacter vibrioides, overall poorly recognizes ␤-lactam substrates and shows an unusual substrate profile compared to other MBLs. In order to understand the structural basis of these properties, the crystal structure of BJP-1 was obtained at 1.4-Å resolution. This revealed significant differences in the conformation and locations of the active-site loops, determining a rather narrow active site and the presence of a unique N-terminal helix bearing Phe-31, whose side chain binds in the active site and represents an obstacle for ␤-lactam substrate binding. In order to probe the potential of sulfonamides (known to inhibit various zinc-dependent enzymes) to bind in the active sites of MBLs, the structure of BJP-1 in complex with 4-nitrobenzenesulfonamide was also obtained (at 1.33-Å resolution), thereby revealing the mode of interaction of these molecules in MBLs. Interestingly, sulfonamide binding resulted in the displacement of the side chain of Phe-31 from its hydrophobic binding pocket, where the benzene ring of the molecule is now found. These data further highlight the structural diversity shown by MBLs but also provide interesting insights in the structure-function relationships of these enzymes. More importantly, we provided the first structural observation of MBL interaction with sulfonamides, which might represent an interesting scaffold for the design of MBL inhibitors.␤-Lactamases are bacterial enzymes that confer resistance to ␤-lactam antibiotics, the most widely used family of anti-infective agents, by hydrolyzing the amide bond of the ␤-lactam ring and thus bear significant clinical relevance (32,41,43). Two structurally and mechanistically distinct families of ␤-lactamases are known: the active-site serine enzymes (Ambler's classes A, C, and D), acting via an acylation-deacylation mechanism, and the metallo-␤-lactamases (MBLs) (Ambler's class B), which require zinc in their active sites and whose catalytic mechanism is less well understood, although several hypotheses have been provided (4, 13, 21, 33). On the basis of sequence homology, three subclasses that also differ by the natures and positions of the residues that constitute the metal binding site(s) have been defined (44).From a structural standpoint, MBLs share a unique fold that was first identified when the structure of the BcII enzyme from Bacillus cereus was solved; this fold consists of a ␤-sandwich flanked by ␣-helices, the metal center being located at the interface of two roughly symmetrical domains (9). Subsequently, many proteins encoded by the genomes of many organisms (from Archaea to mammalians) appeared to share the same typical fold and were grouped in the so-called MBL superfamily, which contains zinc hydrolases that might exhibit a w...