BrabA.11339.a: anomalous diffraction and ligand binding guide towards the elucidation of the function of a 'putative β-lactamase-like protein' from Brucella melitensis

Abstract

The crystal structure of a β-lactamase-like protein from Brucella melitensis was initially solved by SAD phasing from an in-house data set collected on a crystal soaked with iodide. A high-resolution data set was collected at a synchroton at the Se edge wavelength, which also provided an independent source of phasing using a small anomalous signal from metal ions in the active site. Comparisons of anomalous peak heights at various wavelengths allowed the identification of the active-site metal ions as manganese. In the native data set a partially occupied GMP could be identified. When co-crystallized with AMPPNP or GMPPNP, clear density for the hydrolyzed analogs was observed, providing hints to the function of the protein.

Figure 1

Structure of BrabA.11339.a dimer. In the ribbon representation of the crystallo­graphic dimer the β-strands for each protomer are colored slightly differently. The crystallographic dyad runs vertically. Various ions are shown as spheres: Mn²⁺ in magenta, K⁺ in blue and Na⁺ in green. Five strong iodide sites are shown as orange spheres for one protomer. GMP and tartrate are shown as stick models.

Figure 2

Fold comparison. Comparison of the fold of BrabA.11339.a with its structural homologues. 3g1p is the PhnP protein from E. coli, 1zkp is a putative ribonuclease from B. anthracis and 1y44 is ribonuclease Z from B. subtilis. The structural homology is focused around the two β-sheets and helices α4 and α5. Color scheme: Mn, magenta; Zn, cyan; Na, green; K, blue

Figure 3

Dinuclear manganese center. The active site of BrabA.11339.a consists of two manganese ions (magenta), which are bridged by a water molecule (red). The phosphate group of GMP is in close proximity to this activated water molecule. The σ_A-weighted 2F _o − F _c electron density for the high-resolution data set is contoured at 1σ (blue) and the corresponding F _o − F _c electron density is contoured at ±3σ (green/red). The anomalous Fourier electron density is contoured at 10σ (orange). The strong anomalous Fourier density at all three wavelengths indentifies the active-site metal as manganese. For bond distances refer to Table 3 ▶; for anomalous peak heights refer to Table 4 ▶.

Figure 4

AMP-bound and GMP-bound structures. BrabA.11339.a was cocrystallized with AMPPNP and GMPPNP, variants of AMP or GMP that are nonhydrolyzable between the β-­phosphate and γ-phosphate. Left panels, OMIT densities for the AMPPNP (top) and GMPPNP (bottom) cocrystals (see Fig. 3 ▶ for colors). The σ_A-weighted 2F _o − F _c electron density for the high-resolution data set is contoured at 1σ (blue) and the corresponding F _o − F _c electron density is contoured at ±3σ (green/red). The right panels show the refined densities for the models refined with AMP (top) and GMP (bottom), respectively. No density is visible beyond the α-phosphate. It is likely that BrabA.11339.a has hydrolyzed the β-phosphate. The nucleotides are bound in the dimer interface and engage in multiple interactions with both protomers.