Substrate-induced Conformational Changes in the Membrane-embedded IICmtl-domain of the Mannitol Permease from Escherichia coli, EnzymeIImtl, Probed by Tryptophan Phosphorescence Spectroscopy

Abstract

Membrane-bound transport proteins are expected to proceed via different conformational states during the translocation of a solute across the membrane. Tryptophan phosphorescence spectroscopy is one of the most sensitive methods used for detecting conformational changes in proteins. We employed this technique to study substrate-induced conformational changes in the mannitol permease, EnzymeII mtl , of the phosphoenolpyruvate-dependent phosphotransferase system from Escherichia coli. Ten mutants containing a single tryptophan were engineered in the membrane-embedded IIC mtl -domain, harboring the mannitol translocation pathway. The mutants were characterized with respect to steady-state and time-resolved phosphorescence, yielding detailed, site-specific information of the Trp microenvironment and protein conformational homogeneity. The study revealed that the Trp environments vary from apolar, unstructured, and flexible sites to buried, highly homogeneous, rigid peptide cores. The most remarkable example of the latter was observed for position 97, because its long sub-second phosphorescence lifetime and highly structured spectra in both glassy and fluid media imply a well defined and rigid core around the probe that is typical of ␤-sheet-rich structural motifs. The addition of mannitol had a large impact on most of the Trp positions studied. In the case of position 97, mannitol binding induced partial unfolding of the rigid protein core. On the contrary, for residue positions 126, 133, and 147, both steady-state and time-resolved data showed that mannitol binding induces a more ordered and homogeneous structure around these residues. The observations are discussed in context of the current mechanistic and structural model of EII mtl . A phoA fusion study and hydropathy analysis of the IIC mtl -domain resulted in a topology model with three small periplasmic loops, two large cytoplasmic loops, and six putative membrane-spanning helices (6). It has been proposed that both large cytoplasmic loops fold back into the membrane-embedded part of the protein, lining up a hydrophilic pathway for the translocation of the carbohydrate (7). New structural insight on the basis of cysteine-scanning mutagenesis in the first proposed cytoplasmic loop provided evidence for the presence of this loop protruding, at least partly, into the bilayer (8). For the subcloned IIC mtldomain, a two-dimensional projection structure at 5-Å resolution was determined by electron microscopy crystallography (9). Six regions of high density were found, possibly reflecting six membrane-spanning helices.Of the available spectroscopic techniques, Trp phosphorescence spectroscopy is one of the most sensitive approaches used to study changes in protein conformation, due to the extremely slow (radiative) de-excitation rate of the triplet excited state (ϳ0.2 s Ϫ1 ). This makes Trp phosphorescence 10 8 times more sensitive for quenching processes than Trp fluorescence. A conformational change, nearby or more remote from the Trp probe, is expected to induce ...

Bacterial PEP-Dependent Carbohydrate: Phosphotransferase Systems Couple Sensing and Global Control Mechanisms

“…Others were the preferred target for uncoupling mutations. Still other residues were found to be critical for exposing substrate molecules bound in the IIC domain to the phosphorylation site in the IIB domain [12,13,15]. These data provide strong evidence that during this process, a conserved area of the EIIC domain with the bound substrate molecule, and the area of the EIIB domain near the phosphorylated cysteine must be, at least temporarily, in close contact.…”

“…With only few NMR and X-ray crystallography data available for the soluble domains, analysis of the membrane domains relies on indirect data such as sequence alignments, domain complementation, and protein fusion studies [9,10], cysteine crosslinking and accessibility data [12][13][14], tryptophan fluorescence and phosphorescence studies [15], and the analysis of mutants affected in transport, phosphorylation, and in coupling both activities [16][17][18]. According to such data, EIIC(+D) domains from all PTS families comprise at least six typical trans-membrane helices, i.e., α-helices of ≥21 residues length with a hydrophobicity value ≥1.8.…”

Structural insight into the PTS sugar transporter EIIC

“…Furthermore, residues in the N-terminal half of MtlA (the region corresponding from TM1 to TM4 in bcCHBC) were shown to be in close contact with mannitol, the sugar was shown to be located closer to the periplasm, and substantial differences were observed between solvent exposed residues in MtlA and their expected counterparts in the bcChbc structure[69, 70]. Tryptophan phosphorescence spectroscopy indicated that F97 in MtlA (a predicted cytoplasmic loop residue that overlays TM3 in bcChbC) is part of a β-sheet fold[71]. Interestingly, this region was shown to undergo large conformational changes upon ligand binding with partial unfolding around F97, indicating that what appears to be essentially a dimerization domain in bcChbC may play a more extensive role in sugar translocation in MtlA[71].…”

Localization of the Substrate-binding Site in the Homodimeric Mannitol Transporter, EIImtl, of Escherichia coli

“…are predicted to form a TMH and a periplasmic loop (36) or protrude into the membrane from the cytoplasmic side ( Fig. 2 (29,35,38). These results suggest that residues in this part of IIC mtl are directly involved in the mannitol translocation process.…”

“…Location (29,31) and cysteine accessibility studies (35) show that this part of IIC mtl is highly structured. Indeed, in recent topology models these residues The total intensity was assumed as I(t) ϭ ⌺ i ␣ i exp(Ϫt/ i ) with ⌺ i ␣ i ϭ 1 for the condition with mannitol.…”