Protein Cell 2010, 1(8): 771–779 Protein & Cell DOI 10.1007/s13238-010-0085-z RESEARCH ARTICLE Structural basis for prokaryotic calcium- mediated regulation by a Streptomyces coelicolor calcium binding protein * * ✉ ✉ Xiaoyan Zhao1 , Hai Pang1 , Shenglan Wang2, Weihong Zhou3, Keqian Yang2 , Mark Bartlam3 1 Laboratory of Structural Biology, Tsinghua University, Beijing 100084, China 2 Center for Microbial Metabolism and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China 3 Tianjin Key Laboratory of Protein Science, College of Life Sciences, Nankai University, Tianjin 300071, China ✉ Correspondence: [email protected] (K. Yang), [email protected] (M. Bartlam) Received June 7, 2010 Accepted June 10, 2010 ABSTRACT INTRODUCTION The important and diverse regulatory roles of Ca2+ in The important and diverse regulatory roles of Ca2+ in eukaryotes are conveyed by the EF-hand containing eukaryotic cells are mediated by the Ca2+-sensor proteins calmodulin superfamily. However, the calcium-regulatory of the calmodulin superfamily (Strynadka and James, 1989; proteins in prokaryotes are still poorly understood. In Clapham, 1995). These helix-loop-helix EF-hand proteins this study, we report the three-dimensional structure of (Babu et al., 1988) convey the Ca2+ signal through ion- the calcium-binding protein from Streptomyces coelico- induced conformational changes, followed by functional lor, named CabD, which shares low sequence homology changes for the recognition of target molecules (Ikura et al., with other known helix-loop-helix EF-hand proteins. The 1992). The proteins of the calmodulin superfamily can also CabD structure should provide insights into the biologi- interact with metal ions for buffering or transporting roles in cal role of the prokaryotic calcium-binding proteins. The eukaryotes, where the functions of both Ca2+-buffer and unusual structural features of CabD compared with sensor proteins have been well studied (Ikura, 1996). prokaryotic EF-hand proteins and eukaryotic sarcoplas- In contrast, the Ca2+-sensor roles of prokaryotic calcium- mic calcium-binding proteins, including the bending binding proteins are still hypothetical or poorly understood, conformation of the first C-terminal α-helix, unpaired despite repeated reports of the presence of bacterial calcium ligand-binding EF-hands and the lack of the extreme C- binding proteins with functional EF-hand motifs (Onek and terminal loop region, suggest it may have a distinct and Smith, 1992; Norris et al., 1996), and accumulated evidence significant function in calcium-mediated bacterial phy- that Ca2+ ions are involved in a myriad of physiologically siological processes, and provide a structural basis for important bacterial activities, including sporulation, virulence, potential calcium-mediated regulatory roles in prokar- septation, chemotaxis and phosphorylation (Norris et al., yotes. 1991; Michiels et al., 2002). The first prokaryotic calcium binding protein discovered to contain four canonical helix- KEYWORDS calcium-binding protein, crystal structure, loop-helix EF-hand motifs was calerythrin, a 20-kDa acidic Streptomyces coelicolor, calcium-mediated regulation, EF- protein from the high G + C Gram-positive bacterium Saccar- hand opolyspora erythraea (Leadlay et al., 1984; Swan et al., 1987), which is proposed to function as a Ca2+ buffer rather than as a Ca2+ sensor (Cox and Bairoch, 1988). *These authors contributed equally to the work. © Higher Education Press and Springer-Verlag Berlin Heidelberg 2010 771 Protein & Cell Xiaoyan Zhao et al. The structural study of an 18 kDa calcium binding protein RESULTS from the model actinomycete Streptomyces coelicolor A3(2) (GenBank ID: CAC08420), named CabD, should provide Structural overview insights into its potential calcium-mediated role in prokar- yotes, since there is evidence that overexpression of CabD in Two crystal forms were obtained with good diffraction to the filamentous, soil-dwelling, Gram-positive bacteria Genus higher than 2.0 Å resolution. The crystal structure of a Streptomyces essentially affects the aerial mycelium forma- selenomethionyl (Se-Met) derivative in space group tion (unpublished data), which has been established to be P212121 (cell dimensions a = 32.9, b = 51.0, c = 87.0 Å, α = calcium-dependent and calcium-mediated in actinomycetes 90, β = 90, γ = 90), consisting of one protein molecule and 172 (Natsume et al., 1989; Natsume and Marumo, 1992). The low water molecules in the asymmetric unit, was determined by sequence similarity (less than 28%) between CabD and other multi-wavelength anomalous diffraction (MAD) at 1.5 Å prokaryotic EF-hand proteins (Swan et al., 1987; Bylsma et resolution and refined to an Rwork of 19.7 % and an Rfree of al., 1992; Xi et al., 2000; Yang, 2001; Yonekawa et al., 2001; 24.8% (Table 1). 98.7% of the residues in the final structure lie Michiels et al., 2002; Tossavainen et al., 2003), suggests that in the “most favored” regions of the Ramachandran plot by the it has a distinctive and significant functions in physiologic PROCHECK criteria (Laskowski et al., 1993) and 1.3% of processes in bacteria. them within the “additionally allowed” region. Two amino acid CabD is a compact globular protein with a pair of helix-loop- residues, Leu37 and Ile99, were selectively mutated to helix EF-hand motifs forming the N- and C-terminal domains methionine for preparation of a selenomethionyl derivative. of the molecule, and a predominantly hydrophobic core A native structure was also determined from the isomorphous comprising about 20 aromatic residues. CabD is understood Se-Met structure to an Rwork of 21.0% and an Rfree of 24.0% to share significantly high structural similarity with eukaryotic (Table 1). The root mean square deviation of the super- sarcoplasmic calcium binding proteins (SCP) (Cook et al., imposition of the native and Se-Met structure is only 0.23 Å, 1991; Vijay-Kumar and Cook, 1992; Cook et al., 1993), which which means the two structures have identical conformations. are presumed to function as intracellular Ca2+ buffers in the The structure is complete from residue 5 to 169, and residues muscle and neurons of various invertebrates (Hermann and 82–85 are less well defined from the structure due to the poor Cox, 1995). Although the sequence homology is lower than electron density for a small number of residues at the N- 22%, the highest value from NSCP, a SCP from the terminal end and the flexible packing region. sandworm Nereis diversicolor (Vijay-Kumar and Cook, A different crystal form in space group P1 (a = 36.9, b = 1992). This low sequence but high structural similarity with 40.1, c = 52.2 Å, α = 90, β = 90, γ = 90) was crystallized SCPs implies that CabD shares a common Ca2+ buffering role following treatment with 10 mM of the chelating agent EGTA with the SCPs. However, a calcium-mediated regulatory role during purification in an attempt to remove Ca2+ from the has been ascribed to calexcitin B, a member of the neuronal native structure. The structure, however, still has Ca2+ bound, SCPs (Gombos et al., 2001). The locally unique tertiary fold of despite the presence of two molecules per asymmetric unit calexcitin evidently reveals its regulatory role in addition to and an average r.m.s. deviation with the first crystal form of GTPase activity (Erskine et al., 2006). 0.9 Å, which means there were only minor conformational Here we report the first crystal structure of the Strepto- changes around the EF-hands (data not shown). myces coelicolor calcium binding protein determined to 1.5 Å resolution. The three-dimensional structure of CabD reveals Overall structure of CabD several remarkable differences from other typical calcium binding proteins containing functional EF-hand motifs. The The three-dimensional structure of CabD confirms that it is a rarely bending orientation of the first C-terminal helix compact globular molecule with overall dimensions of displaces it from the base of the hydrophobic pocket, spatially approximately 35 Å × 40 Å × 45 Å. The structure includes providing the capability to interact with the putative target eight α-helices (A: residues 6–17, B: 27–41, C: 48–68, D: molecule. Moreover, the abundance of hydrophobic amino 83–91, E: 93–111, F: 121–131, G: 135–145, H: 155–166), acid residues in the vicinity of the third calcium-binding loop encompassing approximately 61% of the structure. Two and the five ligands coordinating the Ca2+ ion probably neighboring α-helices and the connecting loop compose the contribute to the lower capacity to coordinate metal ions and helix-loop-helix EF-hand motif, and the first and last pairs of the local conformational plasticity. Additionally,, the lack of the EF-hands associate closely to form the N-terminal (residues extreme C-terminal loop region, which is ubiquitously present 1–91) and C-terminal (residues 93–170) domains of the in other prokaryotic calcium binding proteins and SCPs, molecule by the interhelical connection (Fig. 1A). EF-hand exposes the buried cavity and renders it more accessible for pairs separated on opposite sides of the molecule are linked target recognition. Our work provides a structural basis for by a bend consisting of only one residue, Lys92, and are understanding the potential calcium-mediated role of prokar- arranged so as to form a pronounced hydrophobic core yotic calcium binding proteins. comprising of about 20 aromatic residues. An electrostatic 772 © Higher Education Press and Springer-Verlag Berlin Heidelberg 2010 Crystal structure of CabD Protein & Cell Table 1 Data collection and refinement statistics for the native and selenomethionyl CalD from Streptomyces coelicolor data collection statistics native Se-Met space group P212121 P212121 unit-cell parameters (Å) a = 32.9, b = 51.0, c = 87.0 a = 33.1, b = 51.1, c = 87.3 resolution range (Å) 50–1.80 (1.91–1.80) 50–1.50 (1.55–1.50) total reflections 91,632 161,520 unique reflections 21,409 (1,683) 23,403 (2,212) a Rmerge (%) 5.4 (33.7) 7.4 (39.6) completeness (%) 98.9 (94.6) 95.4 (92.4) average I/σ (I) 11.9 (2.8) 12.5 (2.5) mean redundancy 4.3 (3.5) 6.9 (6.6) refinement statistics Rwork (%) 19.7 21.0 Rfree (%) 24.8 24.0 No.