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Mutagenesis and functional reconstitution of chlamydial major outer membrane proteins: VS4 domains are not required for pore formation but modify channel function.

ABSTRACT: Chlamidial organisms are obligate intracellular pathogens containing highly antigenic porin-like major outer membrane proteins (MOMPs). MOMP epitopes are of substantial medical interest, and they cluster within four relatively short variable (VS) domains. If MOMPs adopt a beta-barrel fold, like bacterial porins, the VS domains may form extramembranous loops and the conserved regions of the protein may correspond to predicted membrane-located beta-strands. However, molecular studies on native MOMPs have been hampered by the need to culture chlamydiae in eukaryotic host cells and purification and reconstitution remain problematic. In addition, the organisms are difficult to manipulate genetically, and it has also been difficult to functionally reconstitute recombinant MOMPs. To help overcome these problems and improve our understanding of MOMP structure and function, we cloned and expressed C. trachomatis and C. psittaci MOMPs and functionally reconstituted them at the single-channel level. We measured significant functional differences between the two proteins, and by removing and exchanging VS4, we tested the hypothesis that the largest variable domain forms an extramembranous loop that contributes to these differences. Proteins in which VS4 was deleted continued to form functional ion channels, consistent with the idea that the domain forms an extramembranous protein loop and incompatible with models in which it contributes to predicted membrane-located beta-strands. Additionally, the properties of the chimeric proteins strongly suggested that the VS4 domain interacts closely with other regions of the protein to form the channel entrance or vestibule. Our approach can be used to probe structure-function relationships in chlamydial MOMPs and may have implications for the generation of effective antichlamydial vaccines.

SUBMITTER: Hughes ES

PROVIDER: S-EPMC98071 | biostudies-literature | 2001 Mar

REPOSITORIES: biostudies-literature

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Mutagenesis and functional reconstitution of chlamydial major outer membrane proteins: VS4 domains are not required for pore formation but modify channel function.

Hughes E S ES Shaw K M KM Ashley R H RH

Infection and immunity 20010301 3

Chlamidial organisms are obligate intracellular pathogens containing highly antigenic porin-like major outer membrane proteins (MOMPs). MOMP epitopes are of substantial medical interest, and they cluster within four relatively short variable (VS) domains. If MOMPs adopt a beta-barrel fold, like bacterial porins, the VS domains may form extramembranous loops and the conserved regions of the protein may correspond to predicted membrane-located beta-strands. However, molecular studies on native MOM ...[more]

PMID: 11179342

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Project description:Most voltage-gated K(+) currents are relatively insensitive to extracellular Na(+) (Na(+)(o)), but Na(+)(o) potently inhibits outward human ether-a-go-go-related gene (HERG)-encoded K(+) channel current (Numaguchi, H., J.P. Johnson, Jr., C.I. Petersen, and J.R. Balser. 2000. Nat. Neurosci. 3:429-30). We studied wild-type (WT) and mutant HERG currents and used two strategic probes, intracellular Na(+) (Na(+)(i)) and extracellular Ba(2+) (Ba(2+)(o)), to define a site where Na(+)(o) interacts with HERG. Currents were recorded from transfected Chinese hamster ovary (CHO-K1) cells using the whole-cell voltage clamp technique. Inhibition of WT HERG by Na(+)(o) was not strongly dependent on the voltage during activating pulses. Three point mutants in the P-loop region (S624A, S624T, S631A) with intact K(+) selectivity and impaired inactivation each had reduced sensitivity to inhibition by Na(+)(o). Quantitatively similar effects of Na(+)(i) to inhibit HERG current were seen in the WT and S624A channels. As S624A has impaired Na(+)(o) sensitivity, this result suggested that Na(+)(o) and Na(+)(i) act at different sites. Extracellular Ba(2+) (Ba(2+)(o)) blocks K(+) channel pores, and thereby serves as a useful probe of K(+) channel structure. HERG channel inactivation promotes relief of Ba(2+) block (Weerapura, M., S. Nattel, M. Courtemanche, D. Doern, N. Ethier, and T. Hebert. 2000. J. Physiol. 526:265-278). We used this feature of HERG inactivation to distinguish between simple allosteric and pore-occluding models of Na(+)(o) action. A remote allosteric model predicts that Na(+)(o) will speed relief of Ba(2+)(o) block by promoting inactivation. Instead, Na(+)(o) slowed Ba(2+) egress and Ba(2+) relieved Na(+)(o) inhibition, consistent with Na(+)(o) binding to an outer pore site. The apparent affinities of the outer pore for Na(+)(o) and K(+)(o) as measured by slowing of Ba(2+) egress were compatible with competition between the two ions for the channel pore in their physiological concentration ranges. We also examined the role of the HERG closed state in Na(+)(o) inhibition. Na(+)(o) inhibition was inversely related to pulsing frequency in the WT channel, but not in the pore mutant S624A.

Dataset Information

Mutagenesis and functional reconstitution of chlamydial major outer membrane proteins: VS4 domains are not required for pore formation but modify channel function.

Publications

Mutagenesis and functional reconstitution of chlamydial major outer membrane proteins: VS4 domains are not required for pore formation but modify channel function.

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