Edward N. Baker

Structure of human lactoferrin: Crystallographic structure analysis and refinement at 2·8 Å resolution

The structure of human lactoferrin has been refined crystallographically at 2.8 A (1 A = 0.1 nm) resolution using restrained least squares methods. The starting model was derived from a 3.2 A map phased by multiple isomorphous replacement with solvent flattening. Rebuilding during refinement made extensive use of these experimental phases, in combination with phases calculated from the partial model. The present model, which includes 681 of the 691 amino acid residues, two Fe3+, and two CO3(2-), gives an R factor of 0.206 for 17,266 observed reflections between 10 and 2.8 A resolution, with a root-mean-square deviation from standard bond lengths of 0.03 A. As a result of the refinement, two single-residue insertions and one 13-residue deletion have been made in the amino acid sequence, and details of the secondary structure and tertiary interactions have been clarified. The two lobes of the molecule, representing the N-terminal and C-terminal halves, have very similar folding, with a root-mean-square deviation, after superposition, of 1.32 A for 285 out of 330 C alpha atoms; the only major differences being in surface loops. Each lobe is subdivided into two dissimilar alpha/beta domains, one based on a six-stranded mixed beta-sheet, the other on a five-stranded mixed beta-sheet, with the iron site in the interdomain cleft. The two iron sites appear identical at the present resolution. Each iron atom is coordinated to four protein ligands, 2 Tyr, 1 Asp, 1 His, and the specific Co3(2-), which appears to bind to iron in a bidentate mode. The anion occupies a pocket between the iron and two positively charged groups on the protein, an arginine side-chain and the N terminus of helix 5, and may serve to neutralize this positive charge prior to iron binding. A large internal cavity, beyond the Arg side-chain, may account for the binding of larger anions as substitutes for CO3(2-). Residues on the other side of the iron site, near the interdomain crossover strands could provide secondary anion binding sites, and may explain the greater acid-stability of iron binding by lactoferrin, compared with serum transferrin. Interdomain and interlobe interactions, the roles of charged side-chains, heavy-atom binding sites, and the construction of the metal site in relation to the binding of different metals are also discussed.

Pili in Gram-negative and Gram-positive bacteria - structure, assembly and their role in disease

Abstract.Many bacterial species possess long filamentous structures known as pili or fimbriae extending from their surfaces. Despite the diversity in pilus structure and biogenesis, pili in Gram-negative bacteria are typically formed by non-covalent homopolymerization of major pilus subunit proteins (pilins), which generates the pilus shaft. Additional pilins may be added to the fiber and often function as host cell adhesins. Some pili are also involved in biofilm formation, phage transduction, DNA uptake and a special form of bacterial cell movement, known as ‘twitching motility’ In contrast, the more recently discovered pili in Gram-positive bacteria are formed by covalent polymerization of pilin subunits in a process that requires a dedicated sortase enzyme. Minor pilins are added to the fiber and play a major role in host cell colonization.This review gives an overview of the structure, assembly and function of the best-characterized pili of both Gram-negative and Gram-positive bacteria.

Molecular structure, binding properties and dynamics of lactoferrin.

Abstract.Lactoferrin (Lf), a prominent protein in milk, many other secretory fluids and white blood cells, is a monomeric, 80-kDa glycoprotein, with a single polypeptide chain of about 690 amino acid residues. Amino acid sequence relationships place it in the wider transferrin family. Crystallographic analyses of human Lf, and of the Lfs from cow, horse, buffalo and camel, reveal a highly conserved three-dimensional structure, but with differences in detail between species. The molecule is folded into homologous N- and C-terminal lobes, each comprising two domains that enclose a conserved iron binding site. Iron binding and release is accompanied by domain movements that close or open the sites, and is influenced by cooperative interactions between the lobes. Patches of high positive charge on the surface contribute to other binding properties, but the attached glycan chains appear to have little impact on structure and function.

Thiol proteases - comparative studies based on the high-resolution structures of papain and actinidin, and on amino acid sequence information for cathepsins B and H, and stem bromelain.

An accurate three-dimensional structure is known for papain (1.65 A resolution) and actinidin (1.7 A). A detailed comparison of these two structures was performed to determine the effect of amino acid changes on the conformation. It appeared that, despite only 48% identity in their amino acid sequence, different crystallization conditions and different X-ray data collection techniques, their structures are surprisingly similar with a root-mean-square difference of 0.40 A between 76% of the main-chain atoms (differences less than 3 sigma). Insertions and deletions cause larger differences but they alter the conformation over a very limited range of two to three residues only. Conformations of identical side-chains are generally retained to the same extent as the main-chain conformation. If they do change, this is due to a modified local environment. Several examples are described. Spatial positions of hydrogen bonds are conserved to a greater extent than are the specific groups involved. The greatest structural similarity is found for the active site residues of papain and actinidin, for the internal water molecules and for the main-chain conformation of residues in alpha-helices and anti-parallel beta-sheet structure. This was reflected also in the similarity of the temperature factors. It suggests that the secondary structural elements form the skeleton of the molecule and that their interaction is the main factor in directing the fold of the polypeptide chain. Therefore, substitution of residues in the skeleton will, in general, have the most drastic effect on the conformation of the protein molecule. In papain and actinidin, some main-chain-side-chain hydrogen bonds are also strongly conserved and these may determine the folding of non-repetitive parts of the structure. Furthermore, we included primary structure information for three homologous thiol proteases: stem bromelain, and the cathepsins B and H. By combining the three-dimensional structural information for papain and actinidin with sequence homologies and identities, we conclude that the overall folding pattern of the polypeptide chain is grossly the same in all five proteases, and that they utilize the same catalytic mechanism.

Structure of actinidin, after refinement at 1.7 Å resolution

The structure of the sulphydryl protease, actinidin, after refinement at 1.7 A resolution, is described. The positions of most of the 1666 atoms have been determined with an accuracy better than 0.1 A; only two residues (219 and 220) and the side-chain of a third (87) cannot be seen. In addition, the model contains 272 solvent molecules, all taken as water, except one which may be an ammonium ion. Atomic B values give a good indication of the mobility of different parts of the structure. Actinidin has a double domain structure, with one domain mostly helical in its secondary structure, and the other domain built around a twisted β-sheet. The geometry of hydrogen bonds in helices, β-structure and turns has been analysed. All are significantly non-linear, with the angle N-Ĥ…O ~160 °. Carbonyl groups are tilted outwards from the axis of each helix, the tilting apparently unaffected by whether or not additional hydrogen bonds are made (e.g. to water or side-chain atoms). Each domain is folded round a substantial core of non-polar side-chains, but the interface between domains is mostly polar. Interactions across this interface involve a network of eight buried water molecules, the buried carboxyl and amino groups of Glu35, Glu50, Lys181 and Lys17, other polar side-chains and a few hydrophobic groups. One other internal charged side-chain, that of Glu52, is adjacent to a buried solvent molecule, probably an ammonium ion. Other side-chain environments are described. One proline residue has a cis configuration. The sulphydryl group is oxidized, probably to SO2−, with one oxygen atom clearly visible but the other somewhat less certain. The active site geometry is otherwise compatible with the mechanism proposed by Drenth et al. (1975,1976) for papain. The positions of the 272 solvent molecules are described. The best-ordered water molecules are those that are internal (total of 17), in surface pockets, or in the intermolecular contact regions. These generally form three or four hydrogen bonds, two to proton acceptors and one or two to proton donors. Other water molecules make water bridges on the surface, sometimes covering the exposed edges of non-polar groups. Intermolecular contacts involve few protein atoms, but many water molecules.

Stabilizing isopeptide bonds revealed in Gram-positive bacterial pilus structure

Many bacterial pathogens have long, slender pili through which they adhere to host cells. The crystal structure of the major pilin subunit from the Gram-positive human pathogen Streptococcus pyogenes at 2.2 angstroms resolution reveals an extended structure comprising two all-β domains. The molecules associate in columns through the crystal, with each carboxyl terminus adjacent to a conserved lysine of the next molecule. This lysine forms the isopeptide bonds that link the subunits in native pili, validating the relevance of the crystal assembly. Each subunit contains two lysine-asparagine isopeptide bonds generated by an intramolecular reaction, and we find evidence for similar isopeptide bonds in other cell surface proteins of Gram-positive bacteria. The present structure explains the strength and stability of such Gram-positive pili and could facilitate vaccine development.

Crystal structure of the protein disulfide bond isomerase, DsbC, from Escherichia coli.

DsbC is one of five Escherichia coli proteins required for disulfide bond formation and is thought to function as a disulfide bond isomerase during oxidative protein folding in the periplasm. DsbC is a 2 × 23 kDa homodimer and has both protein disulfide isomerase and chaperone activity. We report the 1.9 Å resolution crystal structure of oxidized DsbC where both Cys-X-X-Cys active sites form disulfide bonds. The molecule consists of separate thioredoxin-like domains joined via hinged linker helices to an N-terminal dimerization domain. The hinges allow relative movement of the active sites, and a broad uncharged cleft between them may be involved in peptide binding and DsbC foldase activities.

Crystal structure of hemopexin reveals a novel high-affinity heme site formed between two beta-propeller domains.

The ubiquitous use of heme in animals poses severe biological and chemical challenges. Free heme is toxic to cells and is a potential source of iron for pathogens. For protection, especially in conditions of trauma, inflammation and hemolysis, and to maintain iron homeostasis, a high-affinity binding protein, hemopexin, is required. Hemopexin binds heme with the highest affinity of any known protein, but releases it into cells via specific receptors. The crystal structure of the heme–hemopexin complex reveals a novel heme binding site, formed between two similar four-bladed β-propeller domains and bounded by the interdomain linker. The ligand is bound to two histidine residues in a pocket dominated by aromatic and basic groups. Further stabilization is achieved by the association of the two β-propeller domains, which form an extensive polar interface that includes a cushion of ordered water molecules. We propose mechanisms by which these structural features provide the dual function of heme binding and release.

Dealing with iron: Common structural principles in proteins that transport iron and heme

Iron is essential to life, but poses severe problems because of its toxicity and the insolubility of hydrated ferric ions at neutral pH. In animals, a family of proteins called transferrins are responsible for the sequestration, transport, and distribution of free iron. Comparison of the structure and function of transferrins with a completely unrelated protein hemopexin, which carries out the same function for heme, identifies molecular features that contribute to a successful protein system for iron acquisition, transport, and release. These include a two-domain protein structure with flexible hinges that allow these domains to enclose the bound ligand and provide suitable chemistry for stable binding and an appropriate trigger for release.

A structural framework for understanding the multifunctional character of lactoferrin

Lactoferrin (Lf) is widely distributed, in mammalian milks, other secretory fluids and white blood cells, and its biology is complex. The three-dimensional structure of this important protein was determined in 1987, giving the first atomic view of any member of the transferrin family. This review examines how structural knowledge has contributed to our understanding of Lf function, and what we have yet to understand. The internal structure of Lf is highly conserved, and is dedicated to binding iron, which is sequestered in two almost identical sites, one in each lobe of the molecule. The processes of iron binding and release, and the accompanying conformational changes, are well understood. Some functional properties of Lf derive from this property, both through iron scavenging, and because the structure and dynamics of Lf are altered by its iron status. On the other hand, the external structure (its molecular surface) is much more variable between different Lfs, making it more difficult to identify functionally important sites. One key feature is clear - the cationic N-terminus and associated lactoferricin domain on the N-lobe of Lf. Recent work shows that this region, in addition to its role in antibacterial activity and probable role in DNA binding, is also involved in complex formation with other proteins. Other parts of the surface are more variable and may result in functional differences between the Lfs of different species. Finally, it may be time to re-examine the importance of glycosylation, given the growing evidence that many pathogens depend on binding to glycans for pathogenesis.