Alignment and structure prediction of divergent protein families: periplasmic and outer membrane proteins of bacterial efflux pumps

Johnson, Jason M.; Church, George M.

doi:10.1006/jmbi.1999.2630

Cited by 156 publications

(153 citation statements)

References 112 publications

Supporting

Mentioning

147

Contrasting

Order By: Relevance

“…Adaptors are anchored to the inner membrane by a single transmembrane helix, or by an N-terminal lipid modification. They contain a large periplasmic domain, which is predicted by sequence alignment studies to contain long ␣-helices (13). Whereas the adaptor proteins vary in length and content, the presence of these ␣-helices and a ␤-sandwich domain are thought to be common features (13).…”

mentioning

confidence: 99%

Structure of the periplasmic component of a bacterial drug efflux pump

Higgins

Bokma

Koronakis

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

240

251

View full text Add to dashboard Cite

Multidrug resistance among Gram-negative bacteria is conferred by three-component membrane pumps that expel diverse antibiotics from the cell. These efflux pumps consist of an inner membrane transporter such as the AcrB proton antiporter, an outer membrane exit duct of the TolC family, and a periplasmic protein known as the adaptor. We present the x-ray structure of the MexA adaptor from the human pathogen Pseudomonas aeruginosa. The elongated molecule contains three linearly arranged subdomains; a 47-Å-long ␣-helical hairpin, a lipoyl domain, and a six-stranded ␤-barrel. In the crystal, hairpins of neighboring MexA monomers pack side-by-side to form twisted arcs. We discuss the implications of the packing of molecules within the crystal. On the basis of the structure and packing, we suggest a model for the key periplasmic interaction between the outer membrane channel and the adaptor protein in the assembled drug efflux pump.

show abstract

mentioning

confidence: 99%

Structure of the periplasmic component of a bacterial drug efflux pump

Higgins

Bokma

Koronakis

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

240

251

View full text Add to dashboard Cite

show abstract

“…These three proteins form a channel which permits the extrusion of substrate molecules directly from the cytoplasm or the periplasm into the external medium (20). Several models have been proposed to explain the interactions between the three components of these efflux systems (10,30).…”

mentioning

confidence: 99%

Amino Acid Residues Essential for Function of the MexF Efflux Pump Protein of Pseudomonas aeruginosa

Aires¹,

Pechère²,

Delden³

et al. 2002

Antimicrob Agents Chemother

View full text Add to dashboard Cite

At least four broad-spectrum efflux pumps (Mex) are involved in elevated intrinsic antibiotic resistance as well as in acquired multidrug resistance in Pseudomonas aeruginosa. Substrate specificity of the Mex pumps has been shown to be determined by the cytoplasmic membrane component (MexB, MexD, MexF, and MexY) of the tripartite efflux pump system. Alignment of their amino acid sequences with those of the homologous AcrB and AcrD pump proteins of Escherichia coli showed conservation of five charged amino acid residues located in or next to transmembrane segments (TMS). These residues were mutated in the MexF gene by site-directed mutagenesis and replaced by residues of opposite or neutral charge. MexF proteins containing combined D410A and A411G substitutions located in TMS4 were completely inactive. Similarly, the substitutions E417K (next to TMS4) and K951E (TMS10) also caused loss of activity towards all tested antibiotics. The substitution E349K in TMS2 resulted in a MexF mutant protein which was unable to transport trimethoprim and quinolones but retained partial activity for the transport of chloramphenicol. All mutated MexF proteins were expressed at comparable levels when tested by Western blot analysis. It is concluded that charged residues located in or close to TMS are essential for proper function of MexF.Drug efflux systems endow mammalian cells, yeast, fungi, and bacteria with the ability to become resistant to a variety of chemotherapeutic and antimicrobial agents. Based on their primary amino acid structure, bacterial drug efflux systems can be divided into four major groups: the major facilitator family (14), the staphylococcal (small) multidrug resistance pumps (21), the ATP-binding cassette transporters (3), and the resistance-nodulation-cell division (RND) family (25). Phylogenetically, the latter group can be further subdivided into three clusters which correlate with substrate specificity (26): one containing proteins responsible for heavy metal efflux (CzcA and CnrA), a second involved in secretion of nodulation factors (NolFGH), and the third cluster representing multidrug efflux pumps (Acr, Mex, and Mtr) from various gram-negative bacteria.The Acr, Mex, and Mtr pumps described for Escherichia coli, Pseudomonas aeruginosa, and Neisseria spp., respectively, are composed of three individual proteins. The actual pump component is a 12-transmembrane-segment protein located in the cytoplasmic membrane (6, 7). It is an antiporter deriving the energy for the transport from the proton gradient. The second component is an outer membrane protein which is thought to be connected with the efflux pump by a third component, called a periplasmic link protein or membrane fusion protein (29). These three proteins form a channel which permits the extrusion of substrate molecules directly from the cytoplasm or the periplasm into the external medium (20). Several models have been proposed to explain the interactions between the three components of these efflux systems (10, 30).The Mex pumps of P. aeruginosa...

show abstract

“…The end of the long periplasmic C terminus of Alg44 has similarity to some MFPs of multi-drug efflux systems that export various substances out of the cell. These three-component systems typically consist of a pump located in the cytoplasmic membrane, a periplasmic MFP, and an outer-membrane channel (Johnson & Church, 1999). The membrane pumps located in the cytoplasmic membrane usually belong to the resistance-nodulation division family (RND) or the major facilitator superfamily (MFS) (Johnson & Church, 1999;Nehme et al, 2004).…”

Section: Discussionmentioning

confidence: 99%

“…These three-component systems typically consist of a pump located in the cytoplasmic membrane, a periplasmic MFP, and an outer-membrane channel (Johnson & Church, 1999). The membrane pumps located in the cytoplasmic membrane usually belong to the resistance-nodulation division family (RND) or the major facilitator superfamily (MFS) (Johnson & Church, 1999;Nehme et al, 2004). Multidrug efflux systems are associated with conferring broadrange antibiotic resistance to various Gram-negative bacteria (Elkins & Nikaido, 2003;Tikhonova et al, 2002).…”

Section: Discussionmentioning

confidence: 99%

Membrane topology and roles of Pseudomonas aeruginosa Alg8 and Alg44 in alginate polymerization

2008

View full text Add to dashboard Cite

Mucoid strains of Pseudomonas aeruginosa that overproduce alginate are associated with chronic pulmonary disease (e.g. cystic fibrosis). Mutants defective in one of several periplasmic proteins (AlgKGX) for alginate secretion release alginate fragments due to the activity of an alginate lyase (AlgL) in the periplasm, which cleaves the newly formed polymers. However, mutants defective in Alg8 or Alg44 did not secrete polymer or alginate fragments, suggesting that both these membrane proteins have a role in the polymerization reaction. A model for the membrane topology of Alg8, a glycosyltransferase (GT), was constructed using PhoA fusions. This provided evidence for a large cytoplasmic loop containing the active domains predicted for bGTs such as Alg8 and five transmembrane (TM) domains, one of which resembles a cleavable signal peptide. The C-terminal TM domain of Alg8 was critical for the polymerization reaction in vivo. Alanine substitution mutagenesis showed that all of the predicted active site residues in the widely spaced D, DxD, D, LxxRW motif were required for polymerization activity in vivo, and two of these substitutions also affected Alg8 protein stability. A membrane topology model for Alg44 was also constructed using PhoA fusions, and this showed a central TM domain and predicted an N-terminal TM domain that may be a membrane anchor. An N-terminal PilZ domain in Alg44 for cdi-GMP [bis-(39,59)-cyclic dimeric GMP] binding, which is required for alginate synthesis, was localized to the cytoplasmic loop. The long periplasmic C terminus of Alg44 contains a region similar to membrane fusion proteins (MFPs) of multi-drug efflux systems, which predicts the possibility of its interaction with another protein in this compartment. A Western blot analysis of the outer-membrane porin AlgE showed reduced AlgE levels in the alg44 mutant, whereas expression of Alg44 in trans restored AlgE within the cell. C-terminal truncations of Alg44 as small as 24 amino acids blocked alginate polymerization in vivo, indicating a critical role for the MFP domain. These studies suggest that Alg44 may act as a co-polymerase in concert with Alg8, the major GT, and that both inner-membrane proteins are required in vivo for the polymerization reaction leading to alginate production.

show abstract

Alignment and structure prediction of divergent protein families: periplasmic and outer membrane proteins of bacterial efflux pumps

Cited by 156 publications

References 112 publications

Structure of the periplasmic component of a bacterial drug efflux pump

Structure of the periplasmic component of a bacterial drug efflux pump

Amino Acid Residues Essential for Function of the MexF Efflux Pump Protein of Pseudomonas aeruginosa

Membrane topology and roles of Pseudomonas aeruginosa Alg8 and Alg44 in alginate polymerization

Contact Info

Product

Resources

About