ABSTRACT
BACKGROUND: . The surface protein PsaA of the pathogenic bacterium Streptococcus pneumoniae plays an essential role in its virulence. PsaA is a putative ATP-binding cassette-type (ABC-type) binding protein involved in the uptake of Mn2+ and possibly Zn2+ and is considered to be both a potential drug target and and a candidate vaccine component. RESULTS: . The structure of PsaA has been determined to 2.0 A resolution using X-ray crystallography and is the first structure obtained for an ABC-type binding protein from a Gram-positive organism. The protein consists of two (beta/alpha)4 domains linked together by a single helix. A metal-binding site is formed in the domain interface by the sidechains of His67, His139, Glu205 and Asp280 and is occupied in the structure. CONCLUSIONS: . The structural topology of PsaA is fundamentally different from that of other ABC-type binding proteins determined thus far in that PsaA lacks the characteristic 'hinge peptides' involved in conformational change upon solute uptake and release. In our structure, the metal-binding site is probably occupied by Zn2+. The site seems to be well conserved amongst related receptors from both Gram-positive and Gram-negative bacteria.
Subject(s)
ATP-Binding Cassette Transporters/metabolism , Bacterial Proteins/chemistry , Carrier Proteins , Lipoproteins , Manganese/metabolism , Membrane Transport Proteins , Zinc/metabolism , Adhesins, Bacterial , Amino Acid Sequence , Bacterial Adhesion , Bacterial Proteins/metabolism , Binding Sites , Crystallography, X-Ray , Molecular Sequence Data , Protein Conformation , Sequence Homology, Amino AcidABSTRACT
The putative metal-transporter protein PsaA of Streptococcus pneumoniae is of potential interest both as a vaccine and also as a drug target. The overexpression of the protein in E. coli, and its subsequent purification and crystallization are described. The crystals are rectangular rods and diffract to beyond 2.7 A resolution. The crystal space group is P212121 with unit-cell dimensions a = 59.9, b = 66.5 and c = 69.9 A.
Subject(s)
Bacterial Proteins , Carrier Proteins/chemistry , Lipoproteins/chemistry , Membrane Transport Proteins , Streptococcus pneumoniae/chemistry , Adhesins, Bacterial , Carrier Proteins/biosynthesis , Carrier Proteins/genetics , Carrier Proteins/isolation & purification , Cloning, Molecular , Crystallization , Crystallography, X-Ray , Escherichia coli , Lipoproteins/biosynthesis , Lipoproteins/genetics , Lipoproteins/isolation & purification , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/isolation & purification , Streptococcus pneumoniae/geneticsABSTRACT
We describe here a sub-family of enzymes related both structurally and functionally to N-acetylneuraminate lyase. Two members of this family (N-acetylneuraminate lyase and dihydrodipicolinate synthase) have known three-dimensional structures and we now proceed to show their structural and functional relationship to two further proteins, trans-o-hydroxybenzylidenepyruvate hydratase-aldolase and D-4-deoxy-5-oxoglucarate dehydratase. These enzymes are all thought to involve intermediate Schiff-base formation with their respective substrates. In order to understand the nature of this intermediate, we have determined the three-dimensional structure of N-acetylneuraminate lyase in complex with hydroxypyruvate (a product analogue) and in complex with one of its products (pyruvate). From these structures we deduce the presence of a closely similar Schiff-base forming motif in all members of the N-acetylneuraminate lyase sub-family. A fifth protein, MosA, is also confirmed to be a member of the sub-family although the involvement of an intermediate Schiff-base in its proposed reaction is unclear.
