Daniel Goldberg , '06 and Christopher Kerr, '07
I. Introduction
II. General Structure
III. Leucine-rich Repeat Motif
IV. Water-mediated interactions
V. Structurally Important Residues and Inter-Repeat Interactions
VI. Convex and Concave Sides
VII. The YopM Tetramer
VIII. The Role of the Two Alpha Helices in Translocation and Strengthing Protein Structure
IX. References
Yersinia pestis cytoxin YopM is one of six effector proteins that is associated with the bacterium Yersinia pestis, which is the causative agent of bubonic plague. An effector protein is a type of protein that enables a particular bacterium to evade non-specific immune responses to the bacterium by interfering with the host-cell functions. YopM is a highly acidic protein is essential for virulence of the bacterium.
There are at least six effectors that are injected into the cystol of mammalian cells by Y. pestis. Of the six effectors, YopM is the effector that is least understood. The only activity that is known about YopM is that it binds thrombin, which is an enzyme in blood that facilitates blood clotting. One hypothesis for the function of YopM is that the binding of YopM to thrombin causes a decrease in clotting which would increase bacterial spreading and hinder the body's ability to deliver bactericidal cells to the infection. Another hypothesis is that YopM enters the host cell, associates with parts of the cell until it reaches the nucleus, where it then might change a host's response to the infection. YopM has been observed to accumulate in the cytosol of the cell as well as in the nucleus of the cells, which has not been observed by any of the other effectors.
The general structure of YopM has a modular architecture comprised of 15 tandem copies of a 20/22 "residue leucine repeat motif" . The leucine-rich repeat comprise the central 310 residues in the polypeptide sequence, which are bracketed by 73 residues on the N terminal region and 24 residues on the C terminal region. Three residues in each strand of the inner concave face of the YopM form a beta-sheet, made up of backbone-backbone hydrogen bonding. "A pair of alpha-helices" represent the most canonical aspect of the secondary structure, located within residues 34 through 75 . In addition, these alpha-helices correspond to the signal that communicates to the YopM for translocation into eukaryotic cells. Also included on the YopM is a C-terminal tail and a N-terminal tail. The function of the "N-terminal tail" contains information that targets the protein for secretion from the bacterium and translocation of into mammalian cells . In contrast, little is known about the function of the "C-terminal tail" .
III. Leucine-Rich Repeat Motif
The leucine-rich repeat motif makes up the inner ring structure of the YopM. These leucine-rich repeats are composed of either 20 or 22 residues. The majority of the YopM protein is made up of the leucine-rich repeat containing 20 residues, like leucine-rich repeat 13 . There are only three repeats of the 22 residue type in the YopM protein, like leucine-rich repeat 8 . The presence of leucine-rich repeat is characteristic of bacterial virulence proteins.
IV. Water-mediated interactions
The majority of water interactions occur on the convex side of the protein in order to compensate for the low amount of inter-repeat interactions on this side of the protein . The water interactions on the convex side of the protein might also be needed to allow the protein to be flexible while still maintaining structural integrity. This flexibility is provided by the flexible backbone-water-backbone solvent bridges. In addition, the water-mediated interactions with the backbone do not have a high energetic cost to the protein.
V. Structurally Important Residues and Inter-Repeat Interactions
Highly conserved amino acids found in position 3, 5, 10, 13, 16, and 20 along the leucine-rich repeat include leucine, isoleucine, valine, and alanine, shown here on leucine-rich repeat 13
. The van der Waals interactions that occurs between these residues is the main factor that stabilizes the overall fold of the leucine-rich repeat domain
.
The asparagine residue in position 8 forms strong hydrogen bonds with the main-chain carbonyl group of the isoleucine in position 5
. There are conserved proline residues in positions 14, 17, and 18 that stabilize the extended conformation of the polypeptide chain as well as reinforce the the most flexible region of the leucine-rich repeat backbone
.
The convex side has a semi-regular array of mostly proline and other acidic residues. It is apparent that the concave side of of the protein exposes a variety of amino acid side-chains . It has also been suggested that the concave surface of YopM is used for binding thrombin. The proline residues on the convex side of the protein, like the proline residues on leucine-rich repeat 13, create a thin hydrophobic strip on the mostly negatively charged surface . It is thought that the function of the concave surface of the protein is used for recognition and binding of target proteins.
The tetramer of the superhelix that composes the YopM protein is created by a dimerization between the C-terminal leucine-rich repeat modules of two YopM monomers, like leucine-rich repeat 15 . The interaction between the C-terminal leucine-rich repeat modules buries a large amount of exposed hydrophobic surface on the distal leucine-rich repeat monomers. The alignment of these monomers produces a nearly continuous coil. It is thought that YopM tetramers might form during the process of binding its targets in eukaryotic cells. However, the formation of the tetramers would not be energetically favorable unless physiological conditions in the eukaryotic cell allowed for necessary, specific cellular targets were present.
VIII. Role of the Two Alpha Helices in Translocation and Strengthening Protein Structure
As mentioned above in the general structure of the YopM protein, there are a pair of alpha helices that are located between residues 34-75 . The helices are attached tightly to the proximal end of the leucine-rich repeat. Here are the conserved amino acid residues that are involved in packing the helices and the amino acids involved in binding the helices to the end of the leucine-rich repeat . The role of the two alpha helices may be to facilitate the folding of the adjacent leucine-rich repeat domains by providing a nucleation site of the organization of the proximal domain. This nucleation site could then cause the folding of the other leucine-rich repeat domains in a step-wise fashion. In addition, the two helices form a hydrophilic cap over the hydrophobic core of the proximal leucine-rich repeat. The hydrophilic cap formed by the helices protects the leucine-rich repeat from any type of interaction with a solvent.
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Skrzypek, E. and S.C. Straley. 1996. Interaction between Yersinia pestis YopM protein and human alpha-thrombin. Thrombosis Research. 84: 33-43.
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