Heather M. 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.

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.

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.

Sheep liver cytosolic aldehyde dehydrogenase: the structure reveals the basis for the retinal specificity of class 1 aldehyde dehydrogenases.

BACKGROUND . Enzymes of the aldehyde dehydrogenase family are required for the clearance of potentially toxic aldehydes, and are essential for the production of key metabolic regulators. The cytosolic, or class 1, aldehyde dehydrogenase (ALDH1) of higher vertebrates has an enhanced specificity for all-trans retinal, oxidising it to the powerful differentiation factor all-trans retinoic acid. Thus, ALDH1 is very likely to have a key role in vertebrate development. RESULTS . The three-dimensional structure of sheep ALDH1 has been determined by X-ray crystallography to 2.35 A resolution. The overall tertiary and quaternary structures are very similar to those of bovine mitochondrial ALDH (ALDH2), but there are important differences in the entrance tunnel for the substrate. In the ALDH1 structure, the sidechain of the general base Glu268 is disordered and the NAD+ cofactor binds in two distinct modes. CONCLUSIONS . The submicromolar Km of ALDH1 for all-trans retinal, and its 600-fold enhanced affinity for retinal compared to acetaldehyde, are explained by the size and shape of the substrate entrance tunnel in ALDH1. All-trans retinal fits into the active-site pocket of ALDH1, but not into the pocket of ALDH2. Two helices and one surface loop that line the tunnel are likely to have a key role in defining substrate specificity in the wider ALDH family. The relative sizes of the tunnels also suggest why the bulky alcohol aversive drug disulfiram reacts more rapidly with ALDH1 than ALDH2. The disorder of Glu268 and the observation that NAD+ binds in two distinct modes indicate that flexibility is a key facet of the enzyme reaction mechanism.

Lactoferrin and iron: structural and dynamic aspects of binding and release.

Lactoferrin (Lf) has long been recognized as a member of the transferrin family of proteins and an important regulator of the levels of free iron in the body fluids of mammals. Its ability to bind ferric iron with high affinity (KD∼10−20 M) and to retain it to low pH gives the protein bacteriostatic and antioxidant properties. This ability can be well understood in terms of its three dimensional (3D) structure. The molecule is folded into two homologous lobes (N- and C-lobes) with each lobe binding a single Fe3+ ion in a deep cleft between two domains. The iron sites are highly conserved, and highly favorable for iron binding. Iron binding and release are associated with large conformational changes in which the protein adopts either open or closed states. Comparison of available apolactoferrin structures suggests that iron binding is dependent on the dynamics of the open state. What triggers release of the tightly bound iron, however, and why lactoferrin retains iron to much lower pH than its serum homologue, transferrin, has been the subject of much speculation. Comparisons of structural and functional data on lactoferrins and transferrins now suggest that the key factor comes from cooperative interactions between the two lobes of the molecule, mediated by two α-helices.

Crystal structure of the streptococcal superantigen SPE-C: dimerization and zinc binding suggest a novel mode of interaction with MHC class II molecules

Bacterial superantigens are small proteins that have a very potent stimulatory effect on T lymphocytes through their ability to bind to both MHC class II molecules and T-cell receptors. We have determined the three-dimensional structure of a Streptococcal superantigen, SPE-C, at 2.4 Å resolution. The structure shows that SPE-C has the usual superantigen fold, but that the surface that forms a generic, low-affinity MHC-binding site in other superantigens is here used to create a SPE-C dimer. Instead, MHC class II binding occurs through a zinc binding site that is analogous to a similar site in staphylococcal enterotoxin A. Consideration of the SPE-C dimer suggests a novel mechanism for promotion of MHC aggregation and T-cell activation.

A Randomized Clinical Trial to Reduce Patient Prehospital Delay to Treatment in Acute Coronary Syndrome

Background—Delay from onset of acute coronary syndrome (ACS) symptoms to hospital admission continues to be prolonged. To date, community education campaigns on the topic have had disappointing results. Therefore, we conducted a clinical randomized trial to test whether an intervention tailored specifically for patients with ACS and delivered one-on-one would reduce prehospital delay time. Methods and Results—Participants (n=3522) with documented coronary heart disease were randomized to experimental (n=1777) or control (n=1745) groups. Experimental patients received education and counseling about ACS symptoms and actions required. Patients had a mean age of 67±11 years, and 68% were male. Over the 2 years of follow-up, 565 patients (16.0%) were admitted to an emergency department with ACS symptoms a total of 842 times. Neither median prehospital delay time (experimental, 2.20 versus control, 2.25 hours) nor emergency medical system use (experimental, 63.6% versus control, 66.9%) was different between groups, although experimental patients were more likely than control to call the emergency medical system if the symptoms occurred within the first 6 months following the intervention (P=0.036). Experimental patients were significantly more likely to take aspirin after symptom onset than control patients (experimental, 22.3% versus control, 10.1%, P=0.02). The intervention did not result in an increase in emergency department use (experimental, 14.6% versus control, 17.5%). Conclusions—The education and counseling intervention did not lead to reduced prehospital delay or increased ambulance use. Reducing the time from onset of ACS symptoms to arrival at the hospital continues to be a significant public health challenge. Clinical Trial Registration—clinicaltrials.gov. Identifier NCT00734760.

1.8 A crystal structure of the C-terminal domain of rabbit serum haemopexin.

BACKGROUND Haemopexin is a serum glycoprotein that binds haem reversibly and delivers it to the liver where it is taken up by receptor-mediated endocytosis. Haemopexin has two homologous domains, each having a characteristic fourfold internal sequence repeat. Haemopexin-type domains are also found in other proteins, including the serum adhesion protein vitronectin and various collagenases, in which they mediate protein-protein interactions. RESULTS We have determined the crystal structure of the C-terminal domain of haemopexin at 1.8 A resolution. The domain is folded into four beta-leaflet modules, arranged in succession around a central pseudo-fourfold axis. A funnel-shaped tunnel through the centre of this disc-shaped domain serves as an ion-binding site. CONCLUSIONS A model for haem binding by haemopexin is proposed, utilizing an anion-binding site at the wider end of the central tunnel, together with an associated cleft. This parallels the active-site location in other beta-propeller structures. The capacity to bind both cations and anions, together with the disc shape of the domain, suggests that such domains may be used widely for macromolecular recognition.