PDBsum entry 1c8b

Hydrolase

PDB id: 1c8b

Contents

Protein chains

320 a.a. *

Waters ×126

* Residue conservation analysis

PDB id:

1c8b

Name:

Hydrolase

Title:

Crystal structure of a novel germination protease from spores of bacillus megaterium: structural rearrangements and zymogen activation

Structure:

Spore protease. Chain: a, b. Synonym: germination protease. Engineered: yes

Source:

Bacillus megaterium. Organism_taxid: 1404. Expressed in: escherichia coli. Expression_system_taxid: 562. Other_details: escherichia coli

Biol. unit:

Dimer (from PDB file)

Resolution:

3.00Å R-factor: 0.308 R-free: 0.330

Authors:

K.Ponnuraj,S.Rowland,C.Nessi,P.Setlow,M.J.Jedrzejas

Key ref:

K.Ponnuraj et al. (2000). Crystal structure of a novel germination protease from spores of Bacillus megaterium: structural arrangement and zymogen activation. J Mol Biol, 300, 1. PubMed id: 10864493 DOI: 10.1006/jmbi.2000.3849

Date:

03-May-00 Release date: 03-May-01

Protein chains

P22321  (GPR_BACMQ) -  Germination protease from Priestia megaterium (strain ATCC 12872 / QMB1551)

Seq: 370 a.a.

Struc: 320 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class: E.C.3.4.24.78 - Gpr endopeptidase.

DOI no: 10.1006/jmbi.2000.3849

J Mol Biol 300:1 (2000)

PubMed id: 10864493

Crystal structure of a novel germination protease from spores of Bacillus megaterium: structural arrangement and zymogen activation.

K.Ponnuraj, S.Rowland, C.Nessi, P.Setlow, M.J.Jedrzejas.

ABSTRACT

The DNA in the core of spores of Bacillus species is saturated with a group of small, acid-soluble proteins (SASP) that protect DNA from a variety of harsh treatments and play a major role in spore resistance and long-term spore survival. During spore germination, SASPs are rapidly degraded to amino acids and this degradation is initiated by a sequence-specific protease called germination protease (GPR), which exhibits no obvious mechanistic or amino acid sequence similarity to any known class of proteases. GPR is synthesized during sporulation as an inactive tetrameric zymogen termed P(46), which later autoprocesses to a smaller form termed P(41), which is active only during spore germination. Here, we report the crystal structure of P(46) from Bacillus megaterium at 3.0 A resolution and the fact that P(46) monomer adopts a novel fold. The asymmetric unit contains two P(46) monomers and the functional tetramer is a dimer of dimers, with an approximately 9 A channel in the center of the tetramer. Analysis of the P(46) structure and site-directed mutagenesis studies have provided some insight into the mechanism of zymogen activation as well as the zymogen's lack of activity and the inactivity of P(41) in the mature spore.

Selected figure(s)

Figure 3.
Figure 3. (a) Ribbon diagram of the P[46] monomer. The dotted lines show disordered segments in the structure. (b) Tetrameric structure of P[46]. Monomers A, B, C and D are shown in green, red, blue and magenta, respectively. (c) Topology of the secondary structure of a P[46] monomer. The a and 3[10] helices are represented by rectangles and b strands by arrows. The first and last residue numbers for each element are shown. (a) and (b) were created using the program RIBBONS [Carson 1997]. The structure was solved by automated structure determination program SOLVE [Terwilliger and Berendzen 1999]. SOLVE identified a single solution with seven out of 11 selenium sites per monomer. The overall figure of merit of the MAD phasing was 0.53 for 20.0-3.5 Å resolution, and the overall Z-score of the solution was 36.7. The initial phases from SOLVE were improved using the DM program [Cowtan 1994] with cyclic averaging about the NCS coupled with histogram matching and solvent flattening. Masks for symmetry averaging were drawn from the initial electron density map using QUANTA (Molecular Structure Inc., 1996). The correlation coefficient between NCS related regions was improved from 0.63 to 0.93. The electron density map calculated from the improved phases was immediately interpretable and there was continuous density for large parts of the main chain; in addition, side-chain density for many residues was also visible. Although the general course of the polypeptide backbone was clear, there were three breaks in the loop regions that were at the surface of the protein exposed to the solvent region. Initially, the model was fit as a polyalanine trace. For R[free] calculation [Brunger 1992], 6% of the reflections were set aside. The model improvement began with rigid-body refinement followed by positional refinement with very strict 2-fold non-crystallographic constraint applied between two molecules in the asymmetric unit using the 8.0-3.5 Å pseudonative data at l3. After a few such cycles the protein sequence identification was established with the help of Se sites. Then the model was iteratively refined via constant temperature torsion angle dynamics, positional refinement in CNS [Brunger et al 1998] with each refinement cycle followed by manual rebuilding in QUANTA. During iterative rebuilding, individual residue geometries were monitored with the program PROCHECK [Laskowski et al 1993]. Because of the limited resolution of the data and the presence of NCS, a conservative refinement protocol described by [Kleywegt and Jones 1995] was maintained throughout the course of the refinement. After many cycles of positional and torsion angle dynamics refinement the R[free] dropped from 0.45 to 0.36, yielding a model with an R[cryst] of 0.33 (at 3.5 Å). This model exhibits a good stereochemistry and was used for native data refinement at 3.0 Å. Refinement cycles were accepted only when R[free] diminished. At the final stage of refinement, a few cycles of grouped B-factor refinement were carried out and 63 water molecules were included. The R[free] for the final model is 0.33 with the R[cryst] of 0.308. The final model contains residues 1-30, 40-233, 243-271 and 305-371, and the corresponding 2F[o] - F[c] map is of good quality (Figure 2(c). The refinement statistics are shown in Table 1. No residue lies in the disallowed region of the Ramachandran map; the parameters calculated as indicators of good main-chain and side-chain stereochemistry are all better than the normal for protein structures refined at this resolution, according to PROCHECK. The structure refinement was complex because all the loop regions in the molecule were either highly flexible or disordered. These regions show high B-factors that increase gradually from the ordered part to the flexible part of these regions. The most disordered regions were extended loops and a small stretch of residues at the N and C termini extending into the solvent region. For most of the residues in these regions, the side-chain density was weak or missing and therefore they were truncated to alanine; the residues for which this was done are shown in red in Figure 1. Deviations from NCS were also observed in some parts of the molecule. Any genuine non-crystallographic differences cannot be modeled, as the data does not extend to sufficient resolution to allow refinement without imposing non-crystallographic constraints. Thus, the high R[cryst] value is likely due to the disorder in the structure (51 residues were missing and 78 residues were truncated to alanine) and the possible deviation from the NCS. The structure was evaluated also by the methods of [Branden and Jones 1990 and Hooft et al 1996] utilizing program WHAT_CHECK, and RIBBONS [Carson 1997]. The quality of the P[46] structure was assessed as acceptable by regular standards and was comparable to other protein structures.

Figure 4.
Figure 4. Closer view of (a) the secondary structural elements and (b) the molecular surface of the channel that passes through the center of the P[46] tetramer as well as (c) the environment of the region around the propeptide cleavage site in P[46]. The dimensions of the channel are vert, similar 10 Å × 9 Å × 50 Å. The channel is composed of four sets of residues from the four monomers (shown in green, red, blue and magenta). Note that the channel surface is highly negatively charged. The propeptide cleavage site is located between the core domain and the cap domain in a deep cavity. Part of the propeptide (residues 10-16) is shown in ball and stick model and the arrow indicates the propeptide cleavage site. (a) and (c) were created using the program RIBBONS [Carson 1997] and (b) was created with GRASP [Nicholls et al 1991].

The above figures are reprinted by permission from Elsevier: J Mol Biol (2000, 300, 1-0) copyright 2000.

Figures were selected by an automated process.