MreB (PDB ID: 4CLZ) from Caulobacter crescentus
Created by: Rasia Li
MreB from Caulobacter crescentus (PDB ID: 4CLZ) belongs to the MreB class of proteins found in non-spherical bacteria, including E. coli, T. maritima, B. subtilis, etc. It is considered to be an actin homologue for prokaryotes due to its similarity in tertiary structure and function to eukaryotic actin and other proteins from the actin superfamily (1, 9). Like actin, MreB forms dynamic protofilaments that are necessary to maintain cell shape and polarity. Depletion of MreB from C. crescentus causes the formation of “lemon-shaped” cells and expanded pre-divisional cells with defective cell walls, which lyse after 10 hours. This indicates the importance of MreB in maintaining cell shape as well as cell wall integrity by coordination with synthesis of peptidoglycans (2). MreB is also known to play a role in polar targeting or protein complexes and chromosome segregation for certain organisms (1).
Members of the actin-superfamily, including MreB, are characterized by monomeric units consisting of two domains (I and II) on either side of a nucleotide binding cleft that serves as the active site for ATP hydrolysis. Each domain consists of two subdomains (A and B). Domain II is the most structurally preserved among proteins in the actin superfamily, while domain IB is the most diverse (1).
Monomers of MreB assemble into protofilaments in their ATP-bound state by insertion of subdomain IIA into the cleft between subdomains IB and IIB on the next subunit (3). Upon polymerization, domain IB rotates 21.7° inwards, resulting in a propeller twist that closes the cleft between domains I and II and flattens of one side of the protein to create the inter-protofilament interface. This twist allows for coordination of a water molecule by E140 and T167 for hydrophilic attack on the gamma phosphate of ATP. In the initial monomeric state, the E140 residue is 1 Å away from the nucleotide and therefore cannot coordinate water, making the monomeric form inactive (1). ATP is hydrolyzed to ADP shortly after incorporation into the filament (4).
In C. crescentus, MreB protofilaments are double stranded, with a 5.1 nm subunit repeat and 6.5 nm width, and are antiparallel—unusual compared to other actin-like proteins. Actin and most other actin-like proteins are arranged in parallel fashion with an implicit polarity, causing elongation to occur preferentially at one end. The antiparallel protofilaments in C. crecentus are non-polar and can therefore elongate or shrink from either end. The antiparallel arrangement allows for both filaments to interact with the membrane and with other proteins. The implications of this unique structure are still largely unknown. The antiparallel arrangement is expected to be conserved among bacterial species due to high similarity in MreB structure (1).
The MreB filaments form straight structures in vitro, but develop a spiral structure in vivo that binds to the inner leaflet of the cell membrane along the length of the cell. This spiral structure evolves throughout the cell cycle of C. crecentus with MreB concentrating towards the middle of the cell just before and during cell division (figure 1). Membrane binding occurs via an N-terminal amphipathic helix and a two-residue hydrophobic loop consisting of Phe-102 and Val-103 (3,5). These two structures make MreB difficult to isolate without aggregation. As a result, the N-terminal amphipathic helix was deleted, and the hydrophobic loop residues were mutated (F102S and V103G) to allow for purification of MreB from C. crescentus for crystallization studies (1).
The particular structure shown here is the single-subunit, monomeric, ADP-bound form of MreB. It contains a point mutation of S283D that disrupts the intraprotofilament interface to prevent polymerization, and binds ADP and Mg2+ (1). A separate point mutation can be made at V118D to disrupt the interprotofilament interface to form only single protofilaments. Either of these mutations in vivo results in spherical shape cells, demonstrating that the protofilaments interact with their flat sides to form double filaments necessary for maintaining cell shape (figure 2). The isoelectric point (pI) of this structure is 6.40, and the molecular weight is 36839.28 Da, as calculated by ExPASy, a bioinformatics resource that has a tool used to calculate theoretical isoelectric point and molecular weight of a protein based on its given sequence (6).
MreB has been crystallized with antimicrobial reagent S-(3,4-dichlorobenzyl) isothiourea (A22) and its derivative, MP265. These two drugs are often used to study cell shape, as they produce effects similar to depletion of MreB in bacterial cells. These compounds act as MreB inhibitors by binding to the active site. MP265 binds to E140 and the gamma phosphate of ATP, preventing coordination of water, thereby preventing ATP hydrolysis and blocking the release of the phosphate group. MP265 also prevents formation of double protofilaments by displacing the dimerization helix (Ala-117-Ala-130) and weakening the stability of the protofilament (1).
The PSI-BLAST program was used to find protein with similar primary structure to MreB from C. crescentus. The program calculates an E value based on sequence homology resulting from gaps in which residues exist in one structure but not the other. A lower E value represents fewer gaps and greater total sequence homology, and an E value of less than 0.05 is considered significant for proteins (7). The Dali server operates similarly to find proteins with similar tertiary structure to MreB from C. crescentus. The program uses sum-of-pairs to compare intramolecular distances and calculate a Z-score. A larger Z score represents a greater structural similarity, and a Z score of greater than 2 is considered significant (8).
The structure of MreB from C. crescentus is very similar to MreB from T. maritima (PDB ID: 1JCE) in both primary structure (Z = 41.5) and tertiary structure (E = 10). The structure of MreB from T. maritima consists of the same four domains with IA and IIA having a common fold with a five-stranded β-sheet and three α-helices connected by a helix (9). The domain structure of MreB from C. crescentus follows a similar secondary structure pattern. The antiparallel filaments of C. crescentus would be expected to apply to the filaments in T. maritima as well. MreB from C. crescentus is also very similar in tertiary structure to that of actin from various eukaryotic species, such as D. melanogaster (PDB ID: 3EKS, Z = 32.5), but not in primary structure (E > 0.05). The primary structure more closely resembles Hsp70 (PDB ID: 3FE1), a heat-shock protein that also belongs to the actin superfamily, despite an insertion of 40 residues in subdomain IIA and an additional substrate binding domain (E = 8e-102) (9, 7). The tertiary structure of Hsp70 is also significantly similar (Z = 26.6) (8). The similarity in structure between prokaryotic MreB and eukaryotic actin seem to suggest an evolutionary linkage (9).