Site-Directed Mutagenesis of Loop L3 of Sucrose Porin ScrY Leads to Changes in Substrate Selectivity

Ulmke, Christine; Kreth, Jens; Lengeler, Joseph W.; Welte, Wolfram; Schmid, Kurt Werner

doi:10.1128/jb.181.6.1920-1923.1999

Cited by 20 publications

(6 citation statements)

References 29 publications

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“…To confer sucrose specificity to LamB, two triple mutants were designed based on sequence and structural comparisons between LamB and ScrY, while the design of the double alanine mutant aimed for a channel with a large cross-section. In agreement with earlier results (Luckey and Nikaido, 1980;Ulmke et al, 1999), both translocation assays demonstrate that there is virtually no sucrose permeation through LamB. It has been demonstrated by X-ray structure analysis (Wang et al, 1997) that the fructose moiety of sucrose is indeed too large to translocate through the LamB channel, whereas the glucose moiety inserts partly into the channel constriction and blocks the channel.…”

Section: Discussionsupporting

confidence: 91%

“…The reverse operation, replacement of ScrY porin residues by structurally equivalent LamB residues, has been performed recently (Ulmke et al, 1999). As analyzed in vivo, the ScrY mutants did not exhibit an improved apparent transport K m for maltose.…”

Section: Crystal Structure Of the Triple Mutant Lamby1mentioning

confidence: 99%

“…Recently, the above-mentioned residues have been replaced by their LamB equivalents in ScrY (Ulmke et al, 1999). The in vivo analysis showed that maltose transport was not significantly improved, while sucrose translocation was impaired.…”

Section: Introductionmentioning

confidence: 99%

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Sucrose transport through maltoporin mutants of Escherichia coli

Gelder¹,

Dutzler²,

Dumas³

et al. 2001

Protein Engineering, Design and Selection

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Maltoporin (LamB) and sucrose porin (ScrY) reside in the bacterial outer membrane and facilitate the passive diffusion of maltodextrins and sucrose, respectively. To gain further insight into the determinants of solute specificity, LamB mutants were designed to allow translocation of sucrose, which hardly translocates through wild-type LamB. Three LamB mutants were studied. (a) Based on sequence and structure alignment of LamB with ScrY, two LamB triple mutants were generated (R109D, Y118D,D121F; R109N,Y118D,D121F) to mimic the ScrY constriction. The crystal structure of the first of these mutants was determined to be 3.2 A and showed an increased ScrY-like cross-section except for D109 that protrudes into the channel. (b) Based on this crystal structure a double mutant was generated by truncation of the two residues that obstruct the channel most in LamB (R109A,Y118A). Analysis of liposome swelling and in vivo sugar uptake demonstrated substantial sucrose permeation through all mutants with the double alanine mutant performing best. The triple mutants did not show a well-defined binding site as indicated by sugar-induced ion current noise analysis, which can be explained by remaining steric interference as deduced from the crystal structure. Binding, however, was observed for the double mutant that had the obstructing residues truncated to alanines.

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Section: Discussionsupporting

confidence: 91%

Section: Crystal Structure Of the Triple Mutant Lamby1mentioning

confidence: 99%

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Sucrose transport through maltoporin mutants of Escherichia coli

Gelder¹,

Dutzler²,

Dumas³

et al. 2001

Protein Engineering, Design and Selection

View full text Add to dashboard Cite

show abstract

“…Transplanting these ScrY residues to LamB resulted in a wider channel, allowing transport of the more bulky sucrose molecule, which is not able to permeate through the wild‐type LamB porin (own unpublished data). The opposite was observed when Asp‐201 of ScrY was replaced by the corresponding LamB residue (Ulmke et al ., 1999). This mutation led to a narrowing of the substrate range of ScrY to that resembling LamB.…”

Section: Substrate‐specific Porinsmentioning

confidence: 99%

Structure and function of bacterial outer membrane proteins: barrels in a nutshell

Koebnik¹,

Locher²,

Gelder³

2000

Molecular Microbiology

1,052

886

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The outer membrane protects Gram‐negative bacteria against a harsh environment. At the same time, the embedded proteins fulfil a number of tasks that are crucial to the bacterial cell, such as solute and protein translocation, as well as signal transduction. Unlike membrane proteins from all other sources, integral outer membrane proteins do not consist of transmembrane α‐helices, but instead fold into antiparallel β‐barrels. Over recent years, the atomic structures of several outer membrane proteins, belonging to six families, have been determined. They include the OmpA membrane domain, the OmpX protein, phospholipase A, general porins (OmpF, PhoE), substrate‐specific porins (LamB, ScrY) and the TonB‐dependent iron siderophore transporters FhuA and FepA. These crystallographic studies have yielded invaluable insight into and decisively advanced the understanding of the functions of these intriguing proteins. Our review is aimed at discussing their common principles and peculiarities as well as open questions associated with them.

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“…Protein modelling of the WT allele show that the NTC domain extends from the periplasm inwards into the porin and interacts with the inner loop 3 (L3), which traverses the upper region of the OmpU towards to the extracellular side. Domains analogous to NTC and L3 in other porins contribute to pore size and altered open and close conformation states [37,[62][63][64][65][66][67]. Given our findings, it is likely that the presence of the N-terminal coil contributes to a smaller pore size [37] in the WT allele leading to exclusion of bile molecules in contrast to the sensitive alleles.…”

Section: Discussionmentioning

confidence: 53%

Allelic diversity uncovers protein domains contributing to the emergence of antimicrobial resistance

Grant

López‐Pérez

Almagro‐Moreno

2022

Preprint

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Antimicrobial resistance (AMR) remains a major threat to global health. To date, tractable approaches that decipher how AMR emerges within a bacterial population remain limited. Here, we developed a framework that exploits genetic diversity from environmental bacterial populations to decode emergent phenotypes such as AMR. OmpU, is a porin that makes up to 60% of the outer membrane of Vibrio cholerae, the cholera pathogen. This porin is directly associated with the emergence of the bacterium and confers resistance to numerous host antimicrobials. In this study, we examined naturally occurring allelic variants of OmpU in environmental V. cholerae and established associations that connected genotypic variation with phenotypic outcome. We covered the landscape of gene variability and found that the porin forms two major phylogenetic clusters with striking genetic diversity. We generated 14 isogenic mutant strains, each encoding a unique ompU allele, and found that divergent genotypes lead to convergent antimicrobial resistance profiles. We identified and characterized functional domains in OmpU unique to variants conferring AMR-associated phenotypes. Specifically, we identified four conserved domains that are linked with resistance to bile and host-derived antimicrobial peptides. Mutant strains for these domains exhibit differential susceptibility patterns to these and other antimicrobials. Interestingly, a mutant strain in which we exchanged the four domains of the clinical allele for those of a sensitive strain exhibits a resistance profile closer to a porin deletion mutant. Finally, using phenotypic microarrays, we uncovered novel functions of OmpU and their connection with allelic variability. Our findings highlight the suitability of our approach towards dissecting the specific protein domains associated with the emergence of AMR and can be naturally extended to other bacterial pathogens and biological processes.

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Site-Directed Mutagenesis of Loop L3 of Sucrose Porin ScrY Leads to Changes in Substrate Selectivity

Cited by 20 publications

References 29 publications

Sucrose transport through maltoporin mutants of Escherichia coli

Sucrose transport through maltoporin mutants of Escherichia coli

Structure and function of bacterial outer membrane proteins: barrels in a nutshell

Allelic diversity uncovers protein domains contributing to the emergence of antimicrobial resistance

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