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The periplasmic maltose binding protein, MalE, is a major element in maltose transport and in chemotaxis towards this sugar. Previous genetic analysis of the MalE protein revealed functional domains involved in transport and chemotactic functions. Among them the surface located alpha helix 7, which is part of the C-lobe, one of the two lobes forming the three dimensional structure of MalE. Small deletions in this region abolished maltose transport, although maintaining wild-type affinity and specificity as well as a normal chemoreceptor function. It was suggested that alpha helix 7 may be implicated in interactions between the maltose binding protein and the membrane-bound protein complex (Duplay P, Szmelcman S. 1987. Silent and functional changes in the periplasmic maltose binding protein of Escherichia coli K12. II. Chemotaxis towards maltose. J Mol Biol 194:675-678: Duplay P, Szmelcman S, Bedouelle H, Hofnung M. 1987. Silent and functional changes in the periplasmic maltose binding protein of Escherichia coli K12. I: Transport of maltose. J Mol Biol 194:663-673). In this study, we submitted a region of 14 residues--Asp 207 to Gly 220--encompassing alpha helix 7, to genetic analysis by oligonucleotide mediated random mutagenesis. Out of 127 identified mutations, twelve single and five double mutants with normal affinities towards maltose were selected for further investigation. Two types of mutations were characterized, silent mutations that did not affect maltose transport and mutations that heavily impaired transport kinetics, even thought the maltose binding capacity of the mutant proteins remained normal. Three substitutions at Tyr 210 (Y210S, Y210L, Y210N) drastically reduced maltose transport. One substitution at Ala 213 (A213I) and one substitution at Glu 214 (E214K) also impaired transport. These three identified residues, Tyr 210, Ala 213, and Glu 214, which are constituents of alpha helix 7, therefore seem to play some important role in maltose transport, most probably in a productive interaction between the MalE protein and the membrane bound MalFGK2 complex.

The periplasmic maltose-binding protein (MBP or MalE protein) of Escherichia coli is an essential element in the transport of maltose and maltodextrins and in the chemotaxis towards these sugars. On the basis of previous results suggesting their possible role in the activity and fluorescence of MBP, we have changed independently to alanine each of the eight tryptophan residues as well as asparagine 294, which is conserved among four periplasmic sugar-binding proteins. Five of the tryptophan mutations affected activity. In four cases (substitution of Trp62, Trp230, Trp232 and Trp340), there was a decrease in MBP affinity towards maltose correlated with modifications in transport and chemotaxis. According to the present state of the 2.3 A three-dimensional structure of MBP, all four residues are in the binding site. Residues Trp62 and Trp340 are in the immediate vicinity of the bound substrate and appear to have direct contacts with maltose; this is in agreement with the drastic increases in Kd values (respectively 67 and 300-fold) upon their substitution by alanine residues. The modest increase in Kd (12-fold) observed upon mutation of Trp230 would be compatible with the lesser degree of interaction this residue has with the bound substrate and the idea that it plays an indirect role, presumably by keeping other residues involved directly in binding in their proper orientation. Substitution of Trp232 resulted in a small increase in Kd value (2-fold) in spite of the fact that this residue is the closest to the ligand of the tryptophan residues according to the three-dimensional model. In the fifth case, replacement of Trp158, which is distant from the binding site, strongly reduced the chemotactic response towards maltose without affecting the transport parameters or the sugar-binding activities of the mutant protein. Trp158 may therefore be specifically implicated in the interaction of MBP with the chemotransducer Tar, but this effect is likely to be indirect, since Trp158 is buried in the structure of MBP. Of course, some structural rearrangements could be responsible in part for the effects of these mutations. The remaining four mutations were silent. The corresponding residues (Trp10, Trp94, Trp129 and Asn294) are all distant from the sugar-binding site on the crystallographic model of MBP, which is in agreement with their lack of effect on binding. In addition, our results show that they play no role in the interactions with the other proteins of the maltose transport (MalF, MalG or MalK) or chemotaxis (Tar) systems.(ABSTRACT TRUNCATED AT 400 WORDS)

The 177 N-terminal amino acids of CD4, the receptor of the human immunodeficiency virus (HIV), have been expressed in Escherichia coli as genetic fusions to the periplasmic maltose-binding protein (MalE) from this organism. A large fraction of the hybrid proteins can be released from the periplasm by osmotic shock and purified in one step on a cross-linked amylose column eluted with maltose under mild conditions. One hybrid protein binds HIV envelope protein gp160 and neutralizes the virus in vitro. This provides the first example of the production and one-step purification of an active form of an eukaryotic protein by fusion to MalE. The use of this system for mass screening of CD4 mutants, high-scale production of the hybrid protein for structural studies on CD4, testing antiviral compounds, and therapeutic assays is discussed.

The malE gene encodes the periplasmic maltose-binding protein (MBP). Nineteen mutations that still permit synthesis of stable MBP were generated by random insertion of a BamHI octanucleotide into malE and six additional mutations by in-vitro recombinations between mutant genes. The sequence changes were determined; in most cases the linker insertion is accompanied by a small deletion (30 base-pairs on average). The mutant MBP were studied for export, growth on maltose and maltodextrins, maltose transport and binding, and maltose-induced fluorescence changes. Sixteen mutant MBP (out of 21 studied in detail) were found in the periplasmic space: 12 of them retained a high affinity for maltose, and 10 activity for growth on maltose. The results show that several regions of MBP are dispensable for stability, substrate binding and export. Three regions (residues 207 to 220, 297 to 303 and 364 to 370) may be involved in interactions with the MalF or MalG proteins. A region near the C-terminal end is important for maltose binding. Two regions of the mature protein (residues 18 to 42 and 280 to 296) are required for export to, or solubility in, the periplasm.

We examined the chemotactic behavior of ten Escherichia coli mutants able to synthesize a modified periplasmic maltose-binding protein (MBP) retaining high affinity for maltose. Eight were able to grow on maltose (Mal+), two were not (Mal-). In the capillary assay six out of eight of the Mal+ strains showed an optimal response at the same concentration of maltose as the wild-type strain; the amplitude of the response was strongly reduced in two Mal+ mutants and partially affected in one. The amplitude of the chemotactic response of the two Mal- strains was at least equal to that of the wild type, so that the chemotactic and transport functions of MBP were dissociated in these two cases. We define two regions of the protein (residues 297 to 303 and 364 to 369), that are important both for the chemotactic response and for transport, and one region (residues 207 to 220) that is essential for transport but dispensable for chemotaxis. Interestingly, some regions that were found to be inessential for transport are also dispensable for chemotaxis.

Genetic fusions allowing the expression in E. coli of hybrid proteins between a bacterial periplasmic maltose binding protein (MalE) and the CD4 molecule (the receptor of the HIV virus) have been constructed. One of them has kept most of the properties of each constituent: it is exported, can be purified in one step on an affinity column, interacts with anti-MalE and anti-CD4 antibodies, binds HIV gp 120 protein and inactivates HIV virus in an in vitro test.

A plasmid carrying the malE gene, coding for the periplasmic maltose-binding protein of E. coli, was submitted to random mutagenesis by the insertion of a BamHI linker. About 25% of the clones recovered had acquired a BamHI site in the gene malE. Most of the linker insertions were accompanied by small deletions with an average size of 30 base pairs. Among 21 mutants synthesizing a stable maltose binding protein, 8 were still able to grow on maltose. A preliminary analysis of these mutants indicates that certain regions of the protein may not be essential for maltose transport.