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High-Resolution Structure of Rnase P Protein from Thermotoga Maritima

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High-Resolution Structure of Rnase P Protein from Thermotoga Maritima High-resolution structure of RNase P protein from Thermotoga maritima Alexei V. Kazantsev*, Angelika A. Krivenko*, Daniel J. Harrington†, Richard J. Carter‡, Stephen R. Holbrook‡, Paul D. Adams‡, and Norman R. Pace*§ *Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, CO 80309-0347; †Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CA 94025; and ‡Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720 Contributed by Norman R. Pace, April 30, 2003 The structure of RNase P protein from the hyperthermophilic Structural differences between A- and B-type RNase P RNAs bacterium Thermotoga maritima was determined at 1.2-Å resolu- are substantial. Only Ϸ60% of either kind of the RNA is found tion by using x-ray crystallography. This protein structure is from in the other. The sequence similarities among the RNase P an ancestral-type RNase P and bears remarkable similarity to the proteins are low, with Ϸ20–30% amino acid identity. Nonethe- recently determined structures of RNase P proteins from bacteria less, A- and B-type proteins both can activate the other type of that have the distinct, Bacillus type of RNase P. These two types of RNA in vitro (5). Additionally, the B-type RNAs can comple- protein span the extent of bacterial RNase P diversity, so the results ment the A-type protein in vivo (16). Common features in the generalize the structure of the bacterial RNase P protein. The broad structures of the two types of proteins are expected, therefore, phylogenetic conservation of structure and distribution of poten- to provide insight into the elements that participate in the tial RNA-binding elements in the RNase P proteins indicate that all interaction with and activation of the catalytic RNA. of these homologous proteins bind to their cognate RNAs primarily Structural studies of RNase P proteins have so far been limited by interaction with the phylogenetically conserved core of the to B-type proteins, from the low GϩC Gram-positive bacteria RNA. The protein is found to dimerize through an extensive, Bacillus subtilis (17) and Staphylococcus aureus (18). These well-ordered interface. This dimerization may reflect a mechanism proteins have a high degree of similarity, but are closely related of thermal stability of the protein before assembly with the RNA and so do not provide much comparative perspective. We report moiety of the holoenzyme. here a high-resolution x-ray crystal structure of the RNase P protein from Thermotoga maritima, a hyperthermophilic bacte- Nase P catalyzes the divalent metal-dependent hydrolysis of rium with the A-type RNase P RNA. Ra specific phosphodiester bond in pre-tRNAs, to release the Methods 5Ј precursor sequences and produce the mature 5Ј ends of the Crystallization and Data Collection. tRNAs (see refs. 1–4 for reviews). All RNase P species studied The expression, purification, to date are complex holoenzymes composed of one RNA and at and crystallization of the RNase P protein from T. maritima have least one protein component. The bacterial RNase P typically been described (19). Briefly, the protein was overexpressed in Escherichia coli consists of a large RNA (350–400 nt; 100–130 kDa) and one as a GST fusion, the fusion polypeptide was digested with thrombin to release GST, and RNase P protein was small (Ϸ120 aa; 12–13 kDa) protein subunit. The active site of purified by a combination of denaturing and nondenaturing the bacterial RNase P is contained within the RNA. This is ion-exchange chromatography. Plate-like protein crystals were shown by the catalytic activity of the RNA in the absence of the grown by vapor diffusion from 100 mM NaOAc (pH 4.8–5.2), protein moiety in vitro, at high ionic strength (5). At physiological 12–18% PEG 1,500, 200 mM K SO ,and3mg͞ml protein at ionic strength and in vivo, however, the protein component is 2 4 ambient temperature (23°C). To incorporate an anomalously required for pre-tRNA processing (5, 6). scattering atom into the RNase P protein, an L41M mutation was The role of the bacterial RNase P protein in the RNase P introduced into the fusion expression construct, and the mutant reaction is not entirely clear. The fact that it confers high activity protein was overexpressed in E. coli strain BL21(DE3)pLysS on the RNA at physiological ionic strength suggests that the grown in a selenomethionine-containing medium (20). The protein stabilizes the holoenzyme against electrostatic distortion selenomethionine-substituted mutant protein was purified and (7). In vitro, the protein component of bacterial RNase P has crystallized by vapor diffusion as described for the native version been implicated in increasing the turnover rate of the enzyme of the protein, except 1 mM DTT was added where appropriate (7), possibly by contribution to specific binding of the substrate to reduce oxidation of selenomethionine. Nucleation of the over the product (8). Some mutational, enzymatic, and photoaf- mutant crystal growth was promoted by introducing seed mi- finity crosslinking studies have been interpreted to indicate that crocrystals of native protein. Crystals of the selenomethionine- in the bacterial holoenzyme RNase P protein may form direct Ј substituted mutant protein were highly isomorphous to the contacts with the 5 precursor sequence of the substrate pre- crystals of nonmodified protein, indicating that no significant tRNA (9–11). The structural basis of any such interaction is not structural change was introduced by the selenomethionine sub- known. Moreover, there is no consistent structural model for the stitution. interaction between the protein and the RNase P RNA, despite Before data collection, crystals were transferred for at least 5 several crosslink and footprint studies (11–14). min into a cryoprotectant solution [100 mM NaOAc (pH 5.2), BIOCHEMISTRY Phylogenetic comparative analysis has identified two major 35% PEG 1,500, 20 mM K2SO4, and 1 mM DTT] and flash- structural types of bacterial RNase P RNA (15). Both types have cooled in a 1.8-ml vial of liquid propane that was immediately a homologous core structure that consists of about half the immersed into liquid nitrogen. The crystals then were stored sequence lengths of the RNAs, but about half the sequence of each type of RNA has no homologous counterpart in the other RNA. The ancestral type (A type) is phylogenetically predom- Abbreviations: A type, ancestral type; B type, Bacillus type. inant; most bacterial RNase P RNAs conform to the A type of Data deposition: Atomic coordinates and experimental intensities have been deposited in structure. The Bacillus type (B type) structure is found only in the Protein Data Bank, www.rcsb.org (PDB ID code 1NZ0). the phylogenetic group of low GϩC Gram-positive bacteria. §To whom correspondence should be addressed. E-mail: [email protected]. www.pnas.org͞cgi͞doi͞10.1073͞pnas.0932597100 PNAS ͉ June 24, 2003 ͉ vol. 100 ͉ no. 13 ͉ 7497–7502 Downloaded by guest on September 28, 2021 Table 1. Data collection and processing Data set ␭2 (Peak) ␭1 (Inflection) Space group P21 P21 Unit cell, Å, ° a ϭ 56.23 a ϭ 56.27 b ϭ 64.14 b ϭ 64.17 c ϭ 68.30 c ϭ 68.37 ␣ ϭ ␥ ϭ 90 ␣ ϭ ␥ ϭ 90 ␤ ϭ 102.01 ␤ ϭ 102.01 Wavelength, Å 0.9792 0.9794 Resolution, Å 1.20 (1.35–1.20) 30–1.35 1.20 (1.35–1.20) 30–1.35 Total reflections 774,818 (67,203) 707,615 775,753 (67,666) 708,087 Unique reflections 118,678 (16,982) 101,786 118,822 (16,897) 101,925 Redundancy 6.53 (4.01) 6.94 6.53 (4.00) 6.95 Completeness, % 80.0 (38.7) 97.5 79.8 (38.7) 97.5 deff,Å 1.29 1.29 dopt,Å 1.22 I͞␴(I) 5.3 (1.1) 5.6 5.2 (0.9) 5.8 Rmerge, %* 0.059 (0.565) 0.061 0.061 (0.699) 0.065 † Rr.i.m. 0.070 (0.772) 0.070 0.072 (0.954) 0.077 ‡ Rp.i.m. 0.027 (0.372) 0.026 0.027 (0.456) 0.026 PCV, %§ 8.40 (95.8) 8.50 8.70 (119.5) 9.20 Overall B, Å2 13.688 13.860 *Rmerge ϭ͚hkl͚i͉Ihkl,i Ϫ ͗I͘hkl͉͚͞hkl͚iIhkl,i. † 1/2 Rr.i.m. ϭ͚hkl[N͞(N Ϫ 1)] ͚i͉Ihkl,i Ϫ͗I͘hkl͉͚͞hkl͚iIhkl,i. ‡ 1/2 Rp.i.m. ϭ͚hkl[1͞(N Ϫ 1)] ͚i͉Ihkl,i Ϫ ͗I͘hkl͉͚͞hkl͚iIhkl,i. § 2 1/2 PCV (pooled coefficient of variation) ϭ͚hkl{[1͞(N Ϫ 1)]͚i͉Ihkl,i Ϫ ͗I͘hkl͉ } ͚͞hkl͚iIhkl,i. inside a solid propane ‘‘popsicle’’ immersed in liquid nitrogen for phases (overall figure-of-merit value 92.6%) and resulted in an up to a few weeks. Data were collected from a single crystal at interpretable experimental electron density map. cryogenic temperature (100 K) at beam line 5.0.2, Lawrence Berkeley National Laboratory. To minimize noise in measure- Model Building and Refinement. An initial model of the protein ments of the anomalous differences an inverse-beam strategy content of the asymmetric unit was built automatically with the ͞ was used (21). All data were processed with MOSFLM and merged ARP WARP software package (28), using structural amplitudes ␭ with SCALA programs from the CCP4 suite (22). Data redundancy from the 2 data set and experimental phases extended to 1.50-Å and Rp.i.m. were calculated by using the computer program resolution. Six starting models were averaged and refined by RMERGE (23). The processing statistics are given in Table 1. torsion dynamics. A total of 415 of 472 protein residues in the The completeness and the signal-to-noise ratio of the available asymmetric unit were built in this manner to yield a reasonably ϭ ϭ data deteriorated beyond 1.35 Å.
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