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Role of Conserved Gly-Gly Pairs on the Periplasmic Side of LacY
KTH, School of Engineering Sciences (SCI), Theoretical Physics.ORCID iD: 0000-0002-3364-6647
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2016 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 55, no 31, 4326-4332 p.Article in journal (Refereed) Published
Abstract [en]

On the periplasmic side of LacY, two conserved Gly-Gly pairs in helices II and XI (Gly46 and Gly370, respectively) and helices V and VIII (Gly159 and Gly262, respectively) allow close packing of each helix pair in the outward (periplasmic)-closed conformation. Previous studies demonstrate that replacing one Gly residue in each Gly-Gly pair with Trp leads to opening of the periplasmic cavity with abrogation of transport activity, but an increased rate of galactoside binding. To further investigate the role of the Gly-Gly pairs, 11 double-replacement mutants were constructed for each pair at positions 46 (helix II) and 262 (helix VIII). Replacement with Ala or Ser results in decreased but significant transport activity, while replacements with Thr, Val, Leu, Asn, Gln, Tyr, Trp, Glu, or Lys exhibit very little or no transport. Remarkably, however, the double mutants bind galactoside with affinities 10-20-fold higher than that of the pseudo-WT or WT LacY. Moreover, site-directed alkylation of a periplasmic Cys replacement indicates that the periplasmic cavity becomes readily accessible in the double-replacement mutants. Molecular dynamics simulations with the WT and double-Leu mutant in the inward-open/outward-closed conformation provide support for this interpretation. 

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2016. Vol. 55, no 31, 4326-4332 p.
Keyword [en]
Bins, Close packing, Cys replacement, Double mutants, Molecular dynamics simulations, Transport activity, Molecular dynamics
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:kth:diva-194980DOI: 10.1021/acs.biochem.6b00666ISI: 000384959400009ScopusID: 2-s2.0-84981164268OAI: oai:DiVA.org:kth-194980DiVA: diva2:1045782
Note

QC 20161110

Available from: 2016-11-10 Created: 2016-11-01 Last updated: 2016-11-11Bibliographically approved

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