Colin Blake

Structure of prealbumin: secondary, tertiary and quaternary interactions determined by Fourier refinement at 1.8 A.

Abstract The principal elements of the secondary, tertiary and quaternary structure of the tetrameric human plasma prealbumin molecule have been determined by Fourier refinement of X-ray diffraction data at 1.8 A resolution. The subunit has an extensive β-structure composed of eight strands organised into two four-stranded sheets. There is also one short α-helix. The tertiary structure is largely determined by the association of the two β-sheets. Important contributions to the tertiary structure are made by three tyrosines and one aspartic acid involved in side-chain-main-chain interactions; a buried histidine associated with a group of internal water molecules; and a compact cluster of seven aromatic residues. Quaternary interactions occur at two sets of interfaces closely organised around two of the three molecular 2-fold axes. The exclusive monomer-monomer interface is chiefly concerned with antiparallel hydrogen bond interactions which extend the two four-stranded sheets in the monomers to eight-stranded sheets in the dimer. One of the sheet interactions includes water molecules and tyrosine hydroxyls in the hydrogen bond pattern. The dimers associate through both hydrophilic and hydrophobic interactions at interfaces that involve all four monomers.

The structure of amyloid fibrils by electron microscopy and X-ray diffraction.

Publisher Summary The chapter discusses the structural analysis of amyloid fibrils by electron microscopy and X-ray diffraction. A method to isolate amyloid fibrils from tissue was developed, and such preparations of purified fibrils exhibited the classic staining and birefringent characteristics of crude amyloid samples. The further use of high-resolution electron microscopy, examining ultrathin sections of tissue and isolated material, confirmed the fibrillar form as the characteristic appearance of amyloid in the electron microscope. With the ability to prepare samples of isolated amyloid fibrils it began to be possible to probe the molecular structure of the fibrils using X-ray diffraction. The initial use of this technique produced diffraction patterns that showed that the fibrils were composed of polypeptide chains, extended in the so-called cross-β-conformation. Subsequent analyses by X-ray diffraction on a variety of amyloids and also by NMR analysis have confirmed that the protein chains in all amyloid fibrils are predominantly in the cross-β-conformation. The structures of the soluble, globular forms of several amyloidogenic proteins have been determined by the single crystal X-ray crystallography. The origins of the different amyloidoses appear quite diverse, with some arising from a genetic mutation and others apparently from polypeptide misprocessing or from an unusual accumulation of full-length wild-type protein.

From the globular to the fibrous state: protein structure and structural conversion in amyloid formation

The term ‘amyloid’ was used originally to describe certain deposits found post- mortem in organs and tissues, which gave a positive reaction when stained with iodine (Virchow, 1854). Only later was it realized that the material was in fact predominantly proteinaceous, although it is known to be associated with carbohydrates, particularly glucosoaminoglycans, when obtained from many ex vivo sources. With the increasing precision in the definition of amyloid, initially from its characteristic green birefringence when stained with the dye Congo Red (Missmahl & Hartwig, 1953), and later from its particular appearance under the electron microscope (Cohen & Calkins, 1959) and its X-ray diffraction pattern (Eanes & Glenner, 1968), it has become evident that it is a specific fibrillar protein state, which can also be formed by some proteins when denatured in vitro (Burke & Rougvie, 1972), and by synthetic oligopeptides (Bradbury et al . 1960) that may form amyloid spontaneously when placed in pure aqueous medium (Serpell, 1996). Although these latter may form useful experimental systems for the study of amyloid, its major interest at present is that it is associated with a number of prominent lethal diseases (Benson & Wallace, 1989; Pepys, 1994).

Crystallographic studies of the dynamic properties of lysozyme.

The patterns of atomic displacements in the crystals of hen and human lysozyme derived from independent crystallographic refinement are broadly similar. Analysis of the pattern indicates a close correlation with molecular structure, strongly suggestive of intramolecular motion. The active site of lysozyme is located in a region of high displacement. It is concluded that protein mobility may play a significant part in biological activity and that X-ray crystallography can contribute to its analysis.

Synchrotron X-ray studies suggest that the core of the transthyretin amyloid fibril is a continuous β-sheet helix

Background: Amyloid diseases, which include Alzheimers disease and the transmissible spongiform encephalopathies, are characterized by the extracellular deposition of abnormal protein fibrils derived from soluble precursor proteins. Although different precursors seem to generate similar fibrils, no adequate molecular structure of amyloid fibrils has been produced using modern techniques. Knowledge of the fibril structure is essential to understanding the molecular mechanism of amyloid formation and could lead to the development of agents to inhibit or reverse the process. Results: The structure of amyloid fibrils from patients with familial amyloidotic polyneuropathy (FAP), which are derived from transthyretin (TTR) variants, has been investigated by fibre diffraction methods using synchrotron radiation. For the first time a significant high-angle diffraction pattern has been observed showing meridional reflections out to 2 A resolution. This pattern was fully consistent with the previously reported cross-s structure for the fibril, but also reveals a new large scale fibre repeat of 115 A. We interpret this pattern as that of a repeating unit of 24 s strands, which form a complete helical turn of s sheet about an axis parallel to the fibre axis. This structure has not been observed previously. We have built a model of the protofilament of the FAP amyloid fibril based on this interpretation, composed of four s sheets related by a single helix axis coincident with the fibre axis, and shown that it is consistent with the observed X-ray data. Conclusions: This work suggests that amyloid fibrils have a novel molecular structure consisting of s sheets extended in regular helical twists along the length of the fibre. This implies that the polypeptide chains in the fibers are hydrogen-bonded together along the entire length of the fibers, thereby accounting for their great stability. The proposed structure of the FAP fibril requires a TTR building block that is structurally different from the native tetramer. This is likely to be either a monomer or dimer with reorganized or truncated s sheets, suggesting that amyloid formation may require significant structural change in precursor proteins.

Structure of human plasma prealbumin at 2.5 A resolution: A preliminary report on the polypeptide chain conformation, quaternary structure and thyroxine binding

Abstract A 2.5 A resolution electron density map of human plasma prealbumin has been calculated using three isomorphous derivatives. Interpretation of the map shows that the molecule is a tetramer composed of identical subunits. Each subunit is composed of a single polypeptide chain containing between 124 and 128 residues, which is consistent with the tetramer molecular weight of 54,000. Half the residues in each monomer are organized into two extensive β-sheets each composed of four strands. All the strand interactions are antiparallel, with one exception. The remaining residues are involved in loops of various kinds that link the β-strands together, one of which contains a very short α-helix. The isolated monomer has the shape of a prolate ellipsoid, with the two β-sheets forming a large part of its surface. The monomers are linked into stable dimers by further antiparallel β-sheet interactions involving equivalent strands at the edge of each of the monomers β-sheets. This gives the dimer two β-sheets each composed of eight strands, four of which derive from one monomer and four from the other. The dimers are assembled into the complete tetrameric molecule with equivalent β-sheets from each dimer opposed at the centre of the molecule. These β-sheets are kept at greater than contact distance by a short loop of chain from each monomer that forms the major dimer-dimer interaction. The opposed, but separated, β-sheets, form the surface of a large slot that runs through the centre of the molecule. Low-resolution X-ray analysis of the binding of thyroxine and tri-iodothyronine to prealbumin shows that the hormones are bound in two symmetry-related binding sites located deeply in the central slot.

Structure of horse muscle phosphoglycerate kinase: Some results on the chain conformation, substrate binding and evolution of the molecule from a 3 Å Fourier map☆

Abstract A 3 A resolution Fourier map of the glycolytic enzyme 3-phosphoglycerate kinase from horse muscle, has been calculated using a single isomorphous derivative. The map is interpretable and has been used to define the organization of the molecules single polypeptide chain. The enzyme has about 355 amino acid residues, indicating that the molecular weight is close to 38,000. The molecule is highly structured with more than half its residues organized into 11α-helical segments and two large β-sheet structures. The binuclear structure of the enzyme has been confirmed by the highresolution map. The two lobes or domains are clearly structurally independent and physically separated except in the neck region, which is now found to be composed of two chains running in opposite directions. Each domain is organized around a central β-sheet core, which in domain A (the ADP ATP binding domain) is composed of six parallel strands, while that in domain B has five strands in which three parallel strands are followed by two running in the opposite direction. On either side of these two sheets lie most of the α-helical segments, which have a tendency to be aligned parallel with the strands of the sheets. The enzyme co-factors, Mg-ADP and Mg-ATP, have been located in low-resolution difference maps. They are both located at a binding site on domain A, which consists of a slot for the adenine ring at the extremity of the parallel β-sheet. The metal in the ADP complex has been defined, and it appears to be linked to both the α and β-phosphates and an enzyme side-chain. The phosphate chain of the co-factors is located in the space between domain A and domain B, with the γ-phosphate of ATP near a side-chain from domain B. The position of the γ-phosphate, which is transferred during the enzymatic reaction, can be interpreted as supporting a mechanism involving a phosphoenzyme intermediate and suggesting that the active site of the enzyme is divided between the two domains, with domain A carrying the ADP ATP binding site and domain B the phosphoglycerate binding site and the catalytic site. Most remarkably, the structure of domain A bears a strong resemblance to the common nucleotide binding unit that has been found in four dehydrogenases. The location of the ADP ATP binding site on this unit is in an equivalent position to the NAD binding site in the dehydrogenases. This relationship is discussed.

The refined structure of the quinoprotein methanol dehydrogenase from Methylobacterium extorquens at 1.94 å

BACKGROUND Methanol dehydrogenase (MDH) is a bacterial periplasmic quinoprotein; it has pyrrolo-quinoline quinone (PQQ) as its prosthetic group, requires Ca2+ for activity and uses cytochrome cL as its electron acceptor. Low-resolution structures of MDH have already been determined. RESULTS The structure of the alpha 2 beta 2 tetramer of MDH from Methylobacterium extorquens has now been determined at 1.94 A with an R-factor of 19.85%. CONCLUSIONS The alpha-subunit of MDH has an eight-fold radial symmetry, with its eight beta-sheets stabilized by a novel tryptophan docking motif. The PQQ in the active site is held in place by a coplanar tryptophan and by a novel disulphide ring formed between adjacent cysteines which are bonded by an unusual non-planar trans peptide bond. One of the carbonyl oxygens of PQQ is bonded to the Ca2+, probably facilitating attack on the substrate, and the other carbonyl oxygen is out of the plane of the ring, confirming the presence of the predicted free-radical semiquinone form of the prosthetic group.

An X-ray study of the subunit structure of prealbumin

Abstract Prealbumin is a plasma protein which, in conjunction with another protein, is responsible for the plasma transport of vitamin A. X-ray examination of prealbumin crystals has shown that the protein crystallizes in the orthorhombic system but with such a high degree of pseudo body-centring that the real space group is concealed. This has been found to be a consequence of molecular symmetry in the prealbumin molecule. Examination of the pseudosymmetry in terms of models of the protein molecule has permitted the quaternary structure of prealbumin and the real space group to be defined. The prealbumin molecule appears to be a tetramer with a molecular weight of 54,000 ± 5000, composed of identical or nearly identical subunits arranged tetrahedrally.

The preparation of isomorphous derivatives.

Publisher Summary This chapter discusses the preparation of isomorphous derivatives, which represents the main-research effort in protein structure laboratories at the present time. The chemical problem of combining compounds that contain atoms of high atomic number with crystalline proteins to form suitable derivatives is made more difficult by severely restrictive crystallographic criterion, if isomorphism must be satisfied. However, the applications of isomorphous replacement to fully determined protein structures shows great promise in the determination of protein function and more particularly enzyme function. This chapter explores that it is necessary to include a brief theoretical outline of the fundamental problem of protein crystallography and the method currently used in its solution, the practical aspects are the main concerns of this chapter.