Role of Surface-Exposed Loops ofHaemophilus influenzaeProtein P2 in the Mitogen-Activated Protein Kinase Cascade

Abstract

The outer membrane of gram-negative bacteria contains several proteins, and some of these proteins, the porins, have numerous biological functions in the interaction with the host; porins are involved in the activation of signal transduction pathways and, in particular, in the activation of the Raf/MEK1-MEK2/ mitogen-activated protein kinase (MAPK) cascade. The P2 porin is the most abundant outer membrane protein of Haemophilus influenzae type b. A three-dimensional structural model for P2 was constructed based on the crystal structures of Klebsiella pneumoniae OmpK36 and Escherichia coli PhoE and OmpF. The protein was readily assembled into the ␤-barrel fold characteristic of porins, despite the low sequence identity with the template proteins. The model provides information on the structural features of P2 and insights relevant for prediction of domains corresponding to surface-exposed loops, which could be involved in the activation of signal transduction pathways. To identify the role of surface-exposed loops, a set of synthetic peptides were synthesized according to the proposed model and were assayed for MEK1-MEK2/MAPK pathway activation. Our results show that synthetic peptides corresponding to surface loops of protein P2 are able to activate the MEK1-MEK2/MAPK pathways like the entire protein, while peptides modeled on internal ␤ strands are unable to induce significant phosphorylation of the MEK1-MEK2/MAPK pathways. In particular, the peptides corresponding to loops L5 (Lys206 to Gly219), L6B (Ser239 to Lys253), and L7 (Thr280 to Lys287) activate, as the whole protein, essentially JNK and p38.

Septic Shock by Gram-Negative Infections: Role of Outer Membrane Proteins

“…Synthetic peptides, corresponding to the amino acid sequences of variable loop regions facing the cell exterior and thus more probably involved in the initial interaction with the host cell, proved to be able to activate the MEK1-MEK2/MAPK pathways similarly to the entire protein. In contrast, peptides modelled on internal β-strands were ineffective in inducing phosphorylation of such pathways (Galdiero, 2003c). A speculative scheme of signal transduction pathways involved in porin-mediated responses is depicted in Figure 4.…”

Peptides complementary to the active loop of porin P2 from Haemophilus influenzae modulate its activity

“…With regard to other porins, one large loop (loop L3), folding back into the channel, determines effective pore cross section and consequent molecular exclusion limit, as well as its physiological and conductivity properties. 21 Recently, it has been demonstrated that porin P2 from Hib induces activation of signaling pathways in U937 cells through the MEK1-MEK2/MAPK cascade. 21,23,24 Peptide sequences corresponding to variable loop regions facing the cell exterior are able to activate this cascade and provide mitogen-activated protein kinase (MAPK) pathway activation similar to that of the entire porin.…”

“…Its molecular mass varies between 36 and 42 kDa, and it is present in all strains and functions as a porin. 22 P2 contains 16 anti-parallel β strands across the outer membrane, eight large loops of variable length on the external surface of the bacterial membrane, and eight short periplasmic turns 21 ( Figure 1A). All transmembrane regions are relatively conserved among strains, while considerable heterogeneity exists in loop regions.…”

“…All transmembrane regions are relatively conserved among strains, while considerable heterogeneity exists in loop regions. 21 The β-barrel spans the entire outer membrane, forming a trimer with the three-barrel axes almost mutually parallel and perpendicular to the membrane. With regard to other porins, one large loop (loop L3), folding back into the channel, determines effective pore cross section and consequent molecular exclusion limit, as well as its physiological and conductivity properties.…”

“…20 The importance of surface-exposed loops of porins has been the focus of recent studies. 21 Some of the surface-exposed loops are involved in the recognition of ligands, including small molecule nutrients, agents such as bacteriophages or colicins, and probably eukaryotic target cells for bacterial pathogens.…”