ABSTRACT
We describe here the solution NMR structures of two IgG binding domains with highly homologous sequences but different three-dimensional structures. The proteins, G311 and A219, are derived from the IgG binding domains of their wild-type counterparts, protein G and protein A, respectively. Through a series of site-directed mutations and phage display selections, the sequences of G311 and A219 were designed to converge to a point of high-level sequence identity while keeping their respective wild-type tertiary folds. Structures of both artificially evolved sequences were determined by NMR spectroscopy. The main chain fold of G311 can be superimposed on the wild-type alpha/beta protein G structure with a backbone rmsd of 1.4 A, and the A219 structure can be overlaid on the wild-type three-alpha-helix protein A fold also with a backbone rmsd of 1.4 A. The structure of G311, in particular, accommodates a large number of mutational changes without undergoing a change in the overall fold of the main chain. The structural differences are maintained despite a high level (59%) of sequence identity. These proteins serve as starting points for further experiments that will probe basic concepts of protein folding and conformational switching.
Subject(s)
Immunoglobulin G/chemistry , Nerve Tissue Proteins/chemistry , Protein Folding , Staphylococcal Protein A/chemistry , Amino Acid Sequence , Binding Sites , Crystallography, X-Ray , Magnetic Resonance Spectroscopy , Molecular Sequence Data , Mutation , Protein Structure, Secondary , ThermodynamicsABSTRACT
The solution structure of the 154-residue conserved hypothetical protein HI0004 has been determined using multidimensional heteronuclear NMR spectroscopy. HI0004 has sequence homologs in many organisms ranging from bacteria to humans and is believed to be essential in Haemophilus influenzae, although an exact function has yet to be defined. It has a alpha-beta-alpha sandwich architecture consisting of a central four-stranded beta-sheet with the alpha2-helix packed against one side of the beta-sheet and four alpha-helices (alpha1, alpha3, alpha4, alpha5) on the other side. There is structural homology with the eukaryotic matrix metalloproteases (MMPs), but little sequence similarity except for a conserved region containing three histidines that appears in both the MMPs and throughout the HI0004 family of proteins. The solution structure of HI0004 is compared with the X-ray structure of an Aquifex aeolicus homolog, AQ_1354, which has 36% sequence identity over 148 residues. Despite this level of sequence homology, significant differences exist between the two structures. These differences are described along with possible functional implications of the structures.
Subject(s)
Haemophilus influenzae/metabolism , Viral Proteins/chemistry , Amino Acid Sequence , Bacterial Proteins/chemistry , Crystallography, X-Ray , Databases, Protein , Genes, Essential , Histidine/chemistry , Hydrolases/chemistry , Magnetic Resonance Spectroscopy , Models, Molecular , Molecular Sequence Data , Protein Conformation , Protein Structure, Secondary , Sequence Homology, Amino Acid , Thermodynamics , Zinc/chemistryABSTRACT
The solution structure of the acidic protein HI1450 from Haemophilus influenzae has been determined by NMR spectroscopy. HI1450 has homologues in ten other bacterial species including Escherichia coli, Vibrio cholerae, and Yersinia pestis but there are no functional assignments for any members of the family. Thirty-one of the amino acids in this 107-residue protein are aspartates or glutamates, contributing to an unusually low pI of 3.72. The secondary structure elements are arranged in an alpha-alpha-beta-beta-beta-beta order with the two alpha helices packed against the same side of an anti-parallel four-stranded beta meander. Two large loops, one between beta1 and beta2 and the other between beta2 and beta3 bend almost perpendicularly across the beta-strands in opposite directions on the non-helical side of the beta-sheet to form a conserved hydrophobic cavity. The HI1450 structure has some similarities to the structure of the double-stranded DNA (dsDNA) mimic uracil DNA glycosylase inhibitor (Ugi) including the distribution of surface charges and the position of the hydrophobic cavity. Based on these similarities, as well as having a comparable molecular surface to dsDNA, we propose that HI1450 may function as a dsDNA mimic in order to inhibit or regulate an as yet unidentified dsDNA binding protein.
Subject(s)
Bacterial Proteins/chemistry , DNA/chemistry , Haemophilus influenzae/chemistry , Molecular Mimicry , Nuclear Magnetic Resonance, Biomolecular , Amino Acid Sequence , Bacterial Proteins/metabolism , Hydrophobic and Hydrophilic Interactions , Models, Molecular , Molecular Sequence Data , Protein Structure, Secondary , SolutionsABSTRACT
The three-dimensional structures of Haemophilus influenzae proteins whose biological functions are unknown are being determined as part of a structural genomics project to ask whether structural information can assist in assigning the functions of proteins. The structures of the hypothetical proteins are being used to guide further studies and narrow the field of such studies for ultimately determining protein function. An outline of the structural genomics methodological approach is provided along with summaries of a number of completed and in progress crystallographic and NMR structure determinations. With more than twenty-five structures determined at this point and with many more in various stages of completion, the results are encouraging in that some level of functional understanding can be deduced from experimentally solved structures. In addition to aiding in functional assignment, this effort is identifying a number of possible new targets for drug development.
