Sequence Alignment and Homology Threading Reveals Prokaryotic and Eukaryotic Proteins Similar to Lactose Permease

Abstract

Certain prokaryotic transport proteins similar to the lactose permease of Escherichia coli (LacY) have been identified by BLAST searches from available genomic databanks. These proteins exhibit conservation of amino acid residues that participate in sugar binding and H + translocation in LacY. Homology threading of prokaryotic transporters based on the X-ray structure of LacY (PDB ID: 1PV7) and sequence similarities reveals a common overall fold for sugar transporters belonging to the Major Facilitator Superfamily (MFS) and suggest new targets for study. Evolution-based searches for sequence similarities also identify eukaryotic proteins bearing striking resemblance to MFS sugar transporters. Like LacY, the eukaryotic proteins are predicted to have 12 transmembrane domains (TMDs), and many of the irreplaceable residues for sugar binding and H + translocation in LacY appear to be largely conserved. The overall size of the eukaryotic homologs is about twice that of prokaryotic permeases with longer N and C termini and loops between TMDs III-IV and VI-VII. The human gene encoding protein FLJ20160 (NM_017694) consists of six exons located on more than 60,000 bp of DNA sequences and requires splicing to produce mature mRNA. Cellular localization predictions suggest membrane insertion with possible proteolysis at the N terminus, and expression studies with the human protein FJL20160 demonstrate membrane insertion in both E. coli and Pichia pastoris. Widespread expression of the eukaryotic sugar transport candidates suggests an important role in cellular metabolism, particularly in brain and tumors. Homology is observed in the TMDs of both the eukaryotic and prokaryotic proteins that contain residues involved in sugar binding and H + translocation in LacY.

Sugar binding induces an outward facing conformation of LacY

“…As a result, the highly conserved hydrophobic residues Phe-140, Phe-334, and Tyr-350 (cytoplasmic ends of helices V, X, and XI, respectively) should facilitate closure of the inward-facing hydrophilic cavity by coming into close proximity as predicted (21). As shown in the x-ray structure, the periplasmic side of LacY in the inward-facing conformation is tightly packed and completely closed, denying access of sugar to the binding site from the periplasmic side (Fig.…”

Protonation and sugar binding to LacY

“…The distances between the OH group of Tyr-236 and Arg-302, His-322, or Glu-325 are estimated to be 3-4 Å. It is also noteworthy that remarkable conservation of these residues has been found in many other members of the MFS (37,38). Although the high pK a could be caused by Arg-302 or Tyr-236, it is unlikely that any single residue is responsible for the unique pH dependence of sugar binding.…”

Probing of the rates of alternating access in LacY with Trp fluorescence

“…1 Left), but the two highly conserved residues should come into close contact as a result of sugar binding and closing of the cytoplasmic cavity (41). Therefore, replacement of Phe-140 with Trp and Phe-334 with His should generate a His-Trp pair that exhibits sugar-induced quenching of Trp fluorescence as the cavity closes (Fig.…”

“…Only Trp-151, which is critical for maximum sugar affinity (40), is located in the middle of LacY at the apex of the open hydrophilic cavity in the inward-facing conformation (8)(9)(10). Aromatic residues homologous to Trp-151 in LacY are a common feature in many MFS sugar transporters (41). Experiments with single-Trp-151 LacY demonstrate that sugar binding results in blue shift of the fluorescence spectrum reflecting an increase in the hydrophobicity of the local environment.…”