Evidence That Transmembrane Segment 2 of the Lactose Permease Is Part of a Conformationally Sensitive Interface between the Two Halves of the Protein

Abstract

A conserved motif, GXXX(D/E)(R/K)XG(R/K)(R/K), isfound in a large group of evolutionarily related membrane proteins involved in the transport of small molecules across the membrane. This motif is located within the cytoplasmic side of transmembrane domain 2 (TM-2) and extends through the hydrophilic loop that connects transmembrane domains 2 and 3. The motif is repeated again in the second half of the protein. In a previous study concerning the loop 2/3 motif (Jessen-Marshall, A. E., Paul, N. J., and Brooker, R. J. (1995) J. Biol. Chem. 270, 16251-16257), it was shown that the conserved aspartate at the fifth position in the motif is critical for transport activity since a variety of site-directed mutations were found to greatly diminish the rate of transport. In the current study, two of these mutations, in which the conserved aspartate was changed to threonine or serine, were used as parental strains to isolate second site suppressor mutations that restore transport function. A total of 10 different second site mutations were identified among a screen of 19 independent mutants. One of the suppressors was found within loop 1/2 in which Thr-45 was changed to arginine. Since the conserved aspartate and position 45 are at opposite ends of TM-2, these results suggest that the role of the conserved aspartate residue in loop 2/3 is to influence the topology of TM-2. Surprisingly, the majority of suppressor mutations were found in the second half of the permease. All of these are expected to alter helix topology in either of two ways. Some of the mutations involved residues within transmembrane segments 7 and 11 that produced substantial changes in side chain volume: TM-7 (Cys-234 3 Trp or Phe, Gln-241 3 Leu, and Phe-247 3 Val) and TM-11 (Ser-366 3 Phe). Alternatively, other mutations were highly disruptive substitutions at the ends of transmembrane segments or within hydrophilic loops (Gly-257 3 Asp, Val-367 3 Glu, Ala-369 3 Pro, and a 5-codon insertion into loop 11/12). It is hypothesized that the effects of these suppressor mutations are to alter the helical topologies in the second half of the protein to facilitate a better interaction with the first half. Overall, these results are consistent with a transport model in which TM-2 acts as an important interface between the two halves of the lactose permease. According to our tertiary model, this interaction occurs between TM-2 and TM-11.

Suppressor analysis of mutations in the loop 2-3 motif of lactose permease: evidence that glycine-64 is an important residue for conformational changes

“…Based on evolutionary studies, it seems unlikely that the conserved amino acid residues in this motif, including Gly-64, would be directly involved in substrate recognition, since many other transport proteins containing this motif are known to transport chemically different substrates such as antibiotics, amino acids, and Kreb's cycle intermediates. A more general role in the mechanism of protein motion-conformational change has been suggested as an alternative possibility to explain the conservation of this motif among transport proteins (14).…”

“…This type of approach has been previously conducted on the lactose permease. For example, nonfunctional mutations at the fifth position of the loop 2-3 motif (Asp-68 3 Ser or Thr) were used as parental strains to identify second-site mutations that restored permease function (14). The nature of these mutants yielded insight into the possible role of this conserved motif in promoting conformational changes.…”

The Conserved Asparagine and Arginine Are Essential for Catalysis of Mammalian Adenylyl Cyclase

“…Proline to leucine has been shown to render G protein-coupled ␣-factor receptor to be constitutively active and second site suppressor mutations from other amino acids to proline enhance the functioning of several mutated proteins, i.e. triosephosphate isomerase, F 1 F 0 ATP synthase, and lactose permease (52)(53)(54)(55).…”

A general method for determining helix packing in membrane proteinsin situ: Helices I and II are close to helix VII in the lactose permease ofEscherichia coli

“…(ii) Replacement of Asp-68 at the cytoplasmic end of helix II with Ser, Thr, or Glu abolishes permease activity (36), and several second-site revertants of D68S or D68T exhibit high activity (37). Interestingly, in 7 of 18 revertants, the inactive phenotype is suppressed by a mutation in the cytoplasmic half of helix VII in which Cys-234 is replaced with a bulkier residue (Phe or Trp).…”