Solution structure and function of an essential CMP kinase of Streptococcus pneumoniae

Abstract

Streptococcus pneumoniae is a major human pathogen that causes high mortality and morbidity and has developed resistance to many antibiotics. We show that the gene product from SP1603, identified from S. pneumoniae TIGR4, is a CMP kinase that is essential for bacterial growth. It represents an attractive drug target for the development of a novel antibiotic to overcome the problems of drug resistance development for this organism. Here we describe the three-dimensional solution structure of the S. pneumoniae CMP kinase as determined by NMR spectroscopy. The structure consists of eight alpha-helices and two beta-sheets that fold into the classical core domain, the substrate-binding domain, and the LID domain. The three domains of the protein pack together to form a central cavity for substrate-binding and enzymatic catalysis. The S. pneumoniae CMP kinase resembles the fold of the Escherichia coli homolog. An insertion of one residue is observed at the beta-turn in the substrate-binding domain of the S. pneumoniae CMP kinase when compared with the E. coli homolog. Chemical shift perturbations caused by the binding of CMP, CDP, and ATP revealed that CMP or CDP binds to the junction between the core and substrate-binding domains, whereas ATP binds to the junction between the core and LID domains. From NMR relaxation studies, we determined that the loops in the LID domain are highly mobile. These mobile loops could aid in the closing/opening of the LID domain during enzyme catalysis.

Figure 1.

¹⁵N/¹H HSQC spectrum of the uniformly ¹⁵N-labeled S. pneumoniae CMP kinase recorded at DRX500 NMR spectrometer. The folded cross peaks along the ¹⁵N-dimension are indicated with asterisks.

Figure 2.

Stereoview of the backbone (N, C_α, C′) of 20 superimposed NMR-derived structures of S. pneumoniae CMP kinase (residues 14–236). The residues from the core domain and the substrate-binding domain (residues 16–161 and 208–234) were used here for the superposition. The N and C termini are labeled N and C, respectively.

Figure 3.

Ribbon plot (Carson 1987) depicting the averaged minimized NMR structure of S. pneumoniae CMP kinase (residues 14–236). The α-helices, β-strands, and loops are colored red, green, and gray, respectively.

Figure 4.

Sequence alignment of the CMP kinases from S. pneumoniae and E. coli. The sequences are aligned using the three-dimensional structures of the proteins (Briozzo et al. 1998). The arrows and rectangles indicate the secondary structures of β-strands and α-helices, respectively. The dots in the sequence indicate gaps in the alignment. The rectangular open box indicates the phosphate-binding loop (P-loop).

Figure 5.

Stereoviews of the ribbon plots (Carson 1987) comparing the structures of the different CMP kinases. (A) Comparison of S. pneumoniae CMP kinase (residues 14–236) structure (green) with the free E. coli CMP kinase crystal structure (yellow; Briozzo et al. 1998). (B) Comparison of S. pneumoniae CMP kinase (residues 14–236) structure (green) with the E. coli CMP kinase crystal structure in complex with CDP (red; Briozzo et al. 1998). The structures were superimposed by using 202 pairs of C_α atoms in A and 76 pairs of C_α atoms of the core domains in B. It is noted that 13 residues in the α7 and the loop between α7 and β8 are not determined in the free E. coli CMP kinase crystal structure and that there is an insertion in the β-turn between the β3 and β4 in the S. pneumoniae CMP kinase.

Figure 6.

Ribbon plots depicting the chemical shift perturbations of S. pneumoniae CMP kinase (residues 14–236) by the binding of CMP (A) and ATP (B). The ribbons are color coded on the basis of different degrees of chemical shift perturbations. Magenta indicates where the backbone NHs are broadened beyond detection by the binding of the substrate. Yellow indicates where the chemical shifts are perturbed by ≥0.3 ppm for the backbone ¹³C_α and/or ¹⁵N atoms and/or by ≥0.05 ppm for the H_α and/or NH protons. The side chains with their chemical shifts perturbed by ≥0.3 ppm for the carbon atoms and/or by ≥0.05 ppm for the protons are shown in ball-and-stick mode.

Figure 7.

Plot of the measured heteronuclear ¹⁵N{¹H} NOEs of the backbone amides and their uncertainties at 14.1T magnetic field as a function of residue number. Residues for which no results are shown correspond to the N-terminal His tag, six proline residues, and the overlapped residues in the ¹⁵N/¹H HSQC spectra.