Biomedical subjects

L J Beamer

Publications and source records attributed to L J Beamer.

10 recordsLinked to original sources

PubMed2000

The 1.7 A crystal structure of BPI: a study of how two dissimilar amino acid sequences can adopt the same fold.

We have extended the resolution of the crystal structure of human bactericidal/permeability-increasing protein (BPI) to 1.7 A. BPI has two domains with the same fold, but with little sequence similarity. To understand the similarity in structure of the two domains, we compare the corresponding residue positions in the two domains by the method of 3D-1D profiles. A 3D-1D profile is a string formed by assigning each position in the 3D structure to one of 18 environment classes. The environment classes are defined by the local secondary structure, the area of the residue which is buried from solvent, and the fraction of the area buried by polar atoms. A structural alignment between the two BPI domains was used to compare the 3D-1D environments of structurally equivalent positions. Greater than 31% of the aligned positions have conserved 3D-1D environments, but only 13% have conserved residue identities. Analysis of the 3D-1D environmentally conserved positions helps to identify pairs of residues likely to be important in conserving the fold, regardless of the residue similarity. We find examples of 3D-1D environmentally conserved positions with dissimilar residues which nevertheless play similar structural roles. To generalize our findings, we analyzed four other proteins with similar structures yet dissimilar sequences. Together, these examples show that aligned pairs of dissimilar residues often share similar structural roles, stabilizing dissimilar sequences in the same fold.

Amino Acid Sequence↗

PubMed2000

Crystallization and initial crystallographic analysis of phosphomannomutase/phosphoglucomutase from Pseudomonas aeruginosa.

The enzyme phosphomannomutase/phosphoglucomutase (PMM/PGM) catalyzes the conversion of mannose 6-phosphate to mannose 1-phosphate in the second step of the alginate biosynthetic pathway of Pseudomonas aeruginosa. PMM/PGM has been crystallized by hanging-drop vapor diffusion in space group P2(1)2(1)2(1). Crystals diffract to 1.75 A resolution on a synchrotron X-ray source under cryo-cooling conditions. PMM/PGM substituted with selenomethionine has been purified and crystallizes isomorphously with the native enzyme. Structure determination by MAD phasing is under way. Because of its role in alginate biosynthesis, PMM/PGM is a potential target for therapeutic inhibitors to combat P. aeruginosa infections.

Animals↗

PubMed1999

The three-dimensional structure of human bactericidal/permeability-increasing protein: implications for understanding protein-lipopolysaccharide interactions.

Gram-negative bacterial infections are often complicated by the inflammatory properties of lipopolysaccharides (LPS) on or released from the bacterial outer membrane. When present in the mammalian bloodstream, LPS can trigger a series of pathological changes, sometimes resulting in septic shock. Two related mammalian proteins, bactericidal/permeability-increasing protein (BPI) and lipopolysaccharide-binding protein (LBP), are known to affect the LPS-induced inflammatory response and are, therefore, of clinical interest. The recently determined three-dimensional structure of human BPI provides information on the overall protein fold, domain organization, and conserved regions of these two proteins. In addition, the discovery of two apolar lipid binding pockets in BPI indicates a possible site of interaction with LPS. The BPI structure is a powerful tool for the design of site-directed mutants, peptide mimetics/inhibitors, and BPI/LBP chimeras. These studies should help further define the functions of BPI and LBP, and their mechanism of interaction with LPS.

Blood Bactericidal Activity↗

PubMed1998

The BPI/LBP family of proteins: a structural analysis of conserved regions.

Two related mammalian proteins, bactericidal/permeability-increasing protein (BPI) and lipopolysaccharide-binding protein (LBP), share high-affinity binding to lipopolysaccharide (LPS), a glycolipid found in the outer membrane of gram-negative bacteria. The recently determined crystal structure of human BPI permits a structure/function analysis, presented here, of the conserved regions of these two proteins sequences. In the seven known sequences of BPI and LBP, 102 residues are completely conserved and may be classified in terms of location, side-chain chemistry, and interactions with other residues. We find that the most highly conserved regions lie at the interfaces between the tertiary structural elements that help create two apolar lipid-binding pockets. Most of the conserved polar and charged residues appear to be involved in inter-residue interactions such as H-bonding. However, in both BPI and LBP a subset of conserved residues with positive charge (lysines 42, 48, 92, 95, and 99 of BPI) have no apparent structural role. These residues cluster at the tip of the NH2-terminal domain, and several coincide with residues known to affect LPS binding; thus, it seems likely that these residues make electrostatic interactions with negatively charged groups of LPS. Overall differences in charge and electrostatic potential between BPI and LBP suggest that BPI's bactericidal activity is related to the high positive charge of its NH2-terminal domain. A model of human LBP derived from the BPI structure provides a rational basis for future experiments, such as site-directed mutagenesis and inhibitor design.

Acute-Phase Proteins↗

PubMed1998

Detecting distant relatives of mammalian LPS-binding and lipid transport proteins.

In mammals, a family of four lipid binding proteins has been previously defined that includes two lipopolysaccharide binding proteins and two lipid transfer proteins. The first member of this family to have its three-dimensional structure determined is bactericidal/permeability-increasing protein (BPI). Using both the sequence and structure of BPI, along with recently developed sequence-sequence and sequence-structure similarity search methods, we have identified 13 distant members of the family in a diverse set of eukaryotes, including rat, chicken, Caenorhabditis elegans, and Biomphalaria galbrata. Although the sequence similarity between these 13 new members and any of the 4 original members of the BPI family is well below the "twilight zone," their high sequence-structure compatibility with BPI indicates they are likely to share its fold. These findings broaden the BPI family to include a member found in retina and brain, and suggest that a primitive member may have contained only one of the two similar domains of BPI.

Acute-Phase Proteins↗

PubMed1998

The implications of the structure of the bactericidal/permeability-increasing protein on the lipid-transfer function of the cholesteryl ester transfer protein.

The cholesteryl ester transfer protein (CETP) is evolutionarily related to the bactericidal/permeability-increasing protein (BPI). The recently solved structure of BPI shows an elongated, boomerang-shaped molecule, with two hydrophobic pockets opening to its concave side. These pockets each contain a phospholipid molecule. A model of CETP, based on the recently solved crystal structure of BPI, provides the basis for interpreting functional studies on CETP. In this model, C-terminal residues 461-476, which were shown to be required for neutral lipid transfer between plasma lipoproteins, from an amphipathic helix covering the opening of the N-terminal pocket. A possible lipid-transfer mechanism for CETP, with the initial step involving the disordering of lipids in the lipoprotein surface, followed by the flipping and entry of a lipid molecule into the hydrophobic lipid-binding pocket, is hypothesized in light of structural evidence and recent studies.

Antimicrobial Cationic Peptides↗

PubMed1997

Crystal structure of human BPI and two bound phospholipids at 2.4 angstrom resolution.

Bactericidal/permeability-increasing protein (BPI), a potent antimicrobial protein of 456 residues, binds to and neutralizes lipopolysaccharides from the outer membrane of Gram-negative bacteria. At a resolution of 2.4 angstroms, the crystal structure of human BPI shows a boomerang-shaped molecule formed by two similar domains. Two apolar pockets on the concave surface of the boomerang each bind a molecule of phosphatidylcholine, primarily by interacting with their acyl chains; this suggests that the pockets may also bind the acyl chains of lipopolysaccharide. As a model for the related plasma lipid transfer proteins, BPI illuminates a mechanism of lipid transfer for this protein family.

Amino Acid Sequence↗

PubMed1992

Refined 1.8 A crystal structure of the lambda repressor-operator complex.

The crystal structure of the lambda repressor-operator complex has been refined to an R-factor of 18.9% at 1.8 A resolution. This refinement, using data collected at low temperature, has revealed the structure of the N-terminal arm and shows that the interactions of repressor with the two halves of the pseudo-symmetric operator site are significantly different. The two halves of the complex are most similar near the outer edge of the operator site (in a region where the lambda and 434 repressors make similar contacts), but they become increasingly different toward the center of the operator. There are striking differences near the center of the site where it appears that the arm makes significant contacts to only one half of the DNA site. This suggested a new way of aligning the operator sites in phage lambda. The high resolution structure confirms many of the previously noted features of the complex, but also reveals a number of new protein-DNA contacts. It also gives a better view of the extensive H-bonding networks that couple contacts made by different residues and different regions of the protein, and reveals important new details about the helix-turn-helix (HTH) region, and the positions of many water molecules in the complex.

Bacteriophage lambda↗

PubMed1991

The DNA binding arm of lambda repressor: critical contacts from a flexible region.

Segments of protein that do not adopt a well-ordered conformation in the absence of DNA can still contribute to site-specific recognition of DNA. The first six residues (NH2-Ser1-Thr2-Lys3-Lys4-Lys5-Pro6-) of phage lambda repressor are flexible but are important for site-specific binding. Low-temperature x-ray crystallography and codondirected saturation mutagenesis were used to study the role of this segment. All of the functional sequences have the form [X]1-[X]2-[Lys or Arg]3-[Lys]4-[Lys or Arg]5-[X]6. A high-resolution (1.8 angstrom) crystal structure shows that Lys3 and Lys4 each make multiple hydrogen bonds with guanines and that Lys5 interacts with the phosphate backbone. The symmetry of the complex breaks down near the center of the site, and these results suggest a revision in the traditional alignment of the six lambda operator sites.

Amino Acid Sequence↗

PubMed1990

Conserved residues make similar contacts in two repressor-operator complexes.

Comparison of a lambda repressor-operator complex and a 434 repressor-operator complex reveals that three conserved residues in the helix-turn-helix (HTH) region make similar contacts in each of the crystallographically determined structures. These conserved residues and their interactions with phosphodiester oxygens help establish a frame of reference within which other HTH residues make contacts that are critical for site-specific recognition. Such "positioning contacts" may be important conserved features within families of HTH proteins. In contrast, the structural comparisons appear to rule out any simple "recognition code" at the level of detailed side chain-base pair interactions.

Amino Acid Sequence↗