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Cytotoxin Associated Gene A (CagA) of Helicobacter pylori

Inhibiting Host Kinases by Mimicking Host Substrates

The Gram negative, microaerophilic gastric pathogen Helicobacter pylori infects nearly one in every two persons. H. pylori is the only pathogen that colonizes the stomach, and is linked to duodenal and gastric ulcers and adenocarcinomas, and is listed as a Group 1 carcinogen by the World Health Organization. More than three-fourths of infections are asymptomatic.

A mobile genetic locus called the cag pathogenicity island is coupled to the ability to cause ulceration and the positive correlation with cancer.  This 40kb region is estimated to contain over forty genes, most of which code for a T4SS (Type IV secretion system).  The only known substrate of this T4SS is called CagA, a protein composed of about 1,200 amino acids (depending on the strain), which is injected into the epithelial cells lining the stomach.  CagA is now known to interact with at least ten host proteins, and has been shown to manipulate many host cellular functions, including cytoskeletal function, cell-to-cell adhesion, and intracellular signal transduction. How these myriad activities and interactions promote ulceration and malignancy is still unclear, and mechanistic insight into CagA function is especially lacking.  In particular, the biochemical intractability of the virulence factor has precluded significant mechanistic studies.

CagA that is translocated into host cells is phosphorylated as well as localized to the plasma membrane. The so-called “repeats domain,” a region with a strain-specific number of contiguous repeats of a 30-40 residues, contains the EPIYA amino acid motif and is critical to many of the identified biological effects of CagA.  The EPIYA motif is phosphorylated by Src-family protein kinases and c-Abl, and serves as the basis for the interaction with many host proteins in a phosphotyrosine-dependent fashion.  Several cellular proteins have been identified to bind both phosphorylated CagA (at the repeated EPIYA motifs) as well as other regions in a phosphorylation-independent fashion.  A critical phosphorylation-independent interaction occurs between CagA and the PAR1/MARK family of serine/theronine kinases.  The PAR1/MARK family of kinases, through interactions with the atypical protein kinase C (aPKC), regulate the integrity of tight junctions between cells as well as cell polarity.

We have obtained soluble forms of CagA containing the repeats domain. Recombinant production of these constructs revealed that stability in solution was problematic.  The repeats domain often would show significant signs of proteolysis and degradation.  This problem was solved with the co-expression of one of the host targets, the MARK2 kinase.  We were able to crystallize this complex and solve the structure.  Although the CagA construct in the crystals was 120 amino acids in length (verified by running crystals on a gel), only fourteen were found to be ordered in the structure.  These fourteen residues bind the MARK2 kinase in its substrate binding region, mimicking endogenous substrates, and is reminiscent the PKA (protein kinase A) inhibitor PKI.

This binding induces conformations in the activation loop of MARK2 that resemble closely that seen in the active state for most protein kinases, even though no nucleotide is present in the active site or phosphorylation in the activation loop.  The CagA peptide, therefore, appears to function as a substrate analog that traps the kinase in a state that resembles the activated form, but which is incapable of any kinase activity.  Our data indicate that the peptide is both necessary and sufficient for inhibition of the MARK2 kinase activity, and that key, conserved amino acids in the PAR1 family substrates found in CagA are required for this inhibition as well.

 

D. Nesic, M.C. Miller, Z.T. Quinkert, M. Steine, B.T. Chait, and C.E. Stebbins. (2010) "Helicobacter pylori CagA Inhibits PAR1/MARK Family Kinases by Mimicking Host Substrates." Nat Struct Mol Biol. 2010 Jan;17(1):130-2. PMID: 20015680
[Abstract]  [pdf] [pdb]

 

Dr. Dragana Nesic and Marshall Miller were the lead scientists on the CagA project.