Direct Evidence forSphingomonassp. A1 Periplasmic Proteins as Macromolecule-Binding Proteins Associated with the ABC Transporter: Molecular Insights into Alginate Transport in the Periplasm^,

Abstract

A Gram-negative bacterium, Sphingomonas sp. A1, has a macromolecule (alginate) import system consisting of a pit on the cell surface and an alginate-specific ATP-binding cassette importer in the inner membrane. Transport of alginate from the pit to the ABC importer is probably mediated by two periplasmic binding protein homologues (AlgQ1 and AlgQ2). Here we describe characteristics of binding of AlgQ1 and AlgQ2 to alginate and its oligosaccharides through surface plasmon resonance biosensor analysis, UV absorption difference spectroscopy, and X-ray crystallography. Both AlgQ1 and AlgQ2 were inducibly expressed in the periplasm of alginate-grown cells of strain A1. Biosensor analysis indicated that both proteins specifically bind alginate with a high degree of polymerization (>100) and that dissociation constants for alginate with an average molecular mass of 26 kDa are 2.3 x 10(-)(7) M for AlgQ1 and 1.5 x 10(-)(7) M for AlgQ2. An in vitro ATPase assay using the membrane complex, including the alginate ABC importer, suggested that both alginate-bound forms of AlgQ1 and AlgQ2 are closely associated with the importer. X-ray crystallography showed that AlgQ1 consisted of two domains separated by a deep cleft that binds alginate oligosaccharides through a conformational change in the two domains. These results directly show that alginate-binding proteins play an important role in the efficient transport of alginate macromolecules with different degrees of polymerization in the periplasm.

Superchannel of Bacteria: Biological Significance and New Horizons

“…23) Both values are comparable with those of other periplasmic binding proteins. The alginate-bound forms of both AlgQ1 and AlgQ2 associate closely with the transporter 23) and activate ATP hydrolysis by the ABC protein.…”

“…5,23) AlgQ1 and AlgQ2 resemble each other in primary structure and bind specifically to alginate with K d values of 2:3 Â 10 À7 M and 1:5 Â 10 À7 M respectively. 23) Both values are comparable with those of other periplasmic binding proteins. The alginate-bound forms of both AlgQ1 and AlgQ2 associate closely with the transporter 23) and activate ATP hydrolysis by the ABC protein.…”

“…AlgQ1 and AlgQ2 consist of N-and C-domains with an = -structure, and contain a calcium ion in the Cdomain, 23,24) although the extent of opening of the two domains in ligand-free AlgQ1 is greater than in AlgQ2 (rotation angle, 7 ). Three linker loops (loops [1][2][3] connect the N-and C-domains, and the cleft formed between the two domains is the alginate-binding region, which is larger than that observed in other substratebinding proteins, such as maltose-binding proteins.…”

“…AlgQ1 and AlgQ2 bind to alginate in the deep cleft formed on closing (37 for AlgQ1 and 30 for AlgQ2) of the N-and C-domains, and release the polymer on opening of the two domains. 23,27) On going from the open-cleft, ligand-free structure to the closed structure, there is a concerted shift (about 2.8 Å ) of the Glu396 side chain, which moves up into the cleft as a result of sugar binding. 24,27) This ligand-induced movement of Glu396 might be the trigger for the motion that enables the other domain to participate in ligand binding and ultimately to engulf the bound alginate.…”

“…23,24,27) Positively-charged residues in the active cleft enable alginate-binding proteins to bind preferentially to alginate, which is an acidic polysaccharide. The proteins bind tightly to the non-reducing terminal residues of the alginate molecule through van der Waals contacts and hydrogen bonds 24,27) and deliver the macromolecule to the ABC transporter in the inner membrane.…”