Collagen structure: the Madras triple helix and the current scenario

This year marks the 50th anniversary of the coiled-coil triple helical structure of collagen, first proposed by Ramachandran's group from Madras. The structure is unique among the protein secondary structures in that it requires a very specific tripeptide sequence repeat, with glycine being mandatory at every third position and readily accommodates the imino acids proline/hydroxyproline, at the other two positions. The original structure was postulated to be stabilized by two interchain hydrogen bonds, per tripeptide. Subsequent modeling studies suggested that the triple helix is stabilized by one direct inter chain hydrogen bond as well as water mediated hydrogen bonds. The hydroxyproline residues were also implicated to play an important role in stabilizing the collagen fibres. Several high resolution crystal structures of oligopeptides related to collagen have been determined in the last ten years. Stability of synthetic mimics of collagen has also been extensively studied. These have confirmed the essential correctness of the coiled-coil triple helical structure of collagen, as well as the role of water and hydroxyproline residues, but also indicated additional sequence-dependent features. This review discusses some of these recent results and their implications for collagen fiber formation.     

Accommodation of insertion mutations on the surface and in the interior of staphylococcal nuclease.

Alignment of homologous amino acid sequences reveals that insertion mutations are fairly common in evolution. Hitherto, the structural consequences of insertion mutations on the surface and in the interior of proteins of known structures have received little attention. We report here the high-resolution X-ray crystal structures of 2 site-directed insertion mutants of staphylococcal nuclease. The structure of the first insertion mutant, in which 2 glycine residues were inserted on the protein surface in the amino-terminal beta-strand, has been solved to 1.70 A resolution and refined to a crystallographic R value of 0.182. The inserted residues are accommodated in a special 3-residue beta-bulge. A bridging water molecule in the newly created cavity satisfies the hydrogen bonding requirements of the beta-sheet by forming a bifurcated hydrogen bond to 1 beta-strand, and a single hydrogen bond to the other beta-strand. The second insertion mutant contains a single leucine residue inserted at the end of the third beta-strand. The structure was solved to 2.0 A resolution and refined to a final R value of 0.196. The insertion is accommodated in a register shift that changes the conformation of the flexible loop portion of the molecule, relaxing and widening the omega turn. This structural alteration results in changes in position and coordination of a bound calcium ion important for catalysis. These structures illustrate important differences in how amino acid insertions are accommodated: as localized bulges, and as extensive register shifts.     

Water as a hydrogen bonding bridge between a phenol and imidazole. A simple model for water binding in enzymes

The X-ray crystal structure of the mono-hydrate of 2,2-bis(imidazol-1-ylmethyl)-4-methylphenol has been determined. Three hydrogen bonds hold water very tightly in the crystal, as determined by deuterium solid-state NMR. The hydrogen bond between the phenolic hydroxyl and water appears to have about the same strength as the direct hydrogen bond to imidazole, suggesting that the structure can be a good model for hydrogen bonds that are mediated by a water molecule in enzymes.     

Water as a hydrogen bonding bridge between a phenol and imidazole. A simple model for water binding in enzymes

The X-ray crystal structure of the mono-hydrate of 2,2-bis(imidazol-1-ylmethyl)-4-methylphenol has been determined. Three hydrogen bonds hold water very tightly in the crystal, as determined by deuterium solid-state NMR. The hydrogen bond between the phenolic hydroxyl and water appears to have about the same strength as the direct hydrogen bond to imidazole, suggesting that the structure can be a good model for hydrogen bonds that are mediated by a water molecule in enzymes.     

Fibre and molecular structure of thymidylyl-3′,5′-deoxyadenosine

X-ray diffraction and molecular model building studies of an ordered structure of thymidylyl-3′,5′-deoxyadenosine which gives fibre-type diffraction patterns, are consistent with a seven-residues per turn, left-handed structure in which the adenine of one molecule and the thymine of the next are linked together by Hoogsteen type of hydrogen bonds. The structure thus resembles a macromolecule in which units are linked together by hydrogen bonds and stabilized by base stocking. Both nucleosides in the basic molecule are in the anti conformation and both sugar rings have C3′-endo puckers. The C5′-05′ bond of the deoxyadenosine is trans relative to C4′-C3′ and the conformations about the P-03′ and P-05′ bond are gauche^?, trans.     

High-resolution study of the three-dimensional structure of horse heart metmyoglobin

The three-dimensional structure of horse heart metmyoglobin has been refined to a final R-factor of 15.5% for all observed data in the 6.0 to 1.9 A resolution range. The final model consists of 1242 non-hydrogen protein atoms, 154 water molecules and one sulfate ion. This structure has nearly ideal bonding and bond angle geometry. A Luzzati plot of the variation in R-factor with resolution yields an estimated mean co-ordinate error of 0.18 A. An extensive analysis of the pattern of hydrogen bonds formed in horse heart metmyoglobin has been completed. Over 80% of the polypeptide chain is involved in eight helical segments, of which seven are composed mainly of alpha-helical (3.6(13))-type hydrogen bonds; the remaining helix is composed entirely of 3(10) hydrogen bonds. Altogether, of 102 hydrogen bonds between main-chain atoms only six are not involved in helical structures, and four of these six occur within beta-turns. The majority of water molecules in horse heart metmyoglobin are found in solvent networks that range in size from two to 35 members. The size of water molecule networks can be rationalized on the basis of three factors: the number of hydrogen bonds to the protein surface, the presence of charged side-chain atoms, and the ability to bridge to neighboring molecules in the crystal lattice. Bridging water networks form the dominant intermolecular interactions. The backbone conformation of horse heart metmyoglobin is very similar to sperm whale metmyoglobin, with significant differences in secondary structure occurring only near residues 119 and 120, where residues 120 to 123 in sperm whale form a distorted type I reverse turn and the horse heart protein has a type II turn at residues 119 to 122. Nearly all of the hydrogen bonds between main-chain atoms (occurring mainly in helical regions) are common to both proteins, and more than half of the hydrogen bonds involving side-chain atoms observed in horse heart are also found in sperm whale metmyoglobin. Unlike sperm whale metmyoglobin, the heme iron atom in horse heart metmyoglobin is not significantly displaced from the plane of the heme group.     

Characterization of collagenous and non-collagenous peptides of a glycoprotein isolated from alveoli of patients with alveolar proteinosis.

A glycoprotein of Mr 62 000 was isolated from lung lavage material of patients with alveolar proteinosis. The glycoprotein was found to contain (per molecule) 72 residues of glycine, 5 residues of hydroxyproline, 3 molecules of sialic acid, 4.9 molecules of mannose, 4.0 molecules of galactose, 0.9 molecule of fucose and 7.0 molecules of N-acetylglucosamine. Limited pepsin digestion of the glycoprotein resulted in six peptides, three of which contained hydroxyproline and nearly 30% glycine, and two of which contained all the carbohydrate present in the glycoprotein of Mr 62 000. The three peptides containing hydroxyproline and with high content of glycine contained a repeating -Gly-X-Y-sequence in the peptide chain. Partial amino acid-sequence analyses on the peptides derived from the digestion of the alveolar glycoprotein with various proteolytic enzymes indicate that this glycoprotein is characterized by the presence of alternating collagenous and non-collagenous regions in the same polypeptide chain.     

Unfolding of an alpha-helix in water.

We describe a 1 ns molecular dynamics simulation of an 18-residue peptide (corresponding to a portion of the H helix of myoglobin) in water. The initial helical conformation progressively frays to a more disordered structure, with the loss of internal secondary structure generally proceeding from the C-terminus toward the N-terminus. Although a variety of mechanisms are involved in the breaking of helical hydrogen bonds, the formation of transient turn structures, with i----i + 3 hydrogen bonds, and bifurcated hydrogen-bond structures intermediate between alpha and turn or 3(10) structures is a common motif. In some cases a single water molecule is inserted into an internal hydrogen bond, but it is also common to have several water molecules involved in transient intermediates. Overall, the results provide new information about the detailed mechanisms by which helices are made and broken in aqueous solution.     

Computer building of β-helical polypeptide models

A general method is presented for computing the atomic coordinates of helices in which a dipeptide is the repeating unit. The method will generate both single- and double-stranded model helices having idealized bond lengths and angles, and any arbitrary, user-specified, pitch and number of residues per turn. The variation of inter- and intrastrand hydrogen bonds with pitch and number of residues per turn can thus be examined. An application of the method is the construction of a β-helix having pitch of 6.3 Å per turn and 4.85 residues per turn, a model which can pack nicely into the unit cell of crystals of cation-bound gramicidin A.     

The crystal structure of the alpha-cellobiose.2 NaI.2 H(2)O complex in the context of related structures and conformational analysis

The crystal structure of beta-D-glucopyranosyl-(1-->4)-alpha-D-glucopyranose (alpha-cellobiose) in a complex with water and NaI was determined with Mo K(alpha) radiation at 150 K to R=0.027. The space group is P2(1) and unit cell dimensions are a=9.0188, b=12.2536, c=10.9016 A, beta=97.162 degrees. There are no direct hydrogen bonds among cellobiose molecules, and the usual intramolecular hydrogen bond between O-3 and O-5' is replaced by a bridge involving Na+, O-3, O-5', and O-6'. Both Na+ have sixfold coordination. One I(-) accepts six donor hydroxyl groups and three C-H***I(-) hydrogen bonds. The other accepts three hydroxyls, one Na+, and five C-H***I(-) hydrogen bonds. Linkage torsion angles phi(O-5) and psi(C-5) are -73.6 and -105.3 degrees, respectively (phi(H)=47.1 degrees and psi(H)=14.6 degrees ), probably induced by the Na+ bridge. This conformation is in a separate cluster in phi,psi space from most similar linkages. Both C-6-O-H and C-6'-O-H are gg, while the C-6'-O-H groups from molecules not in the cluster have gt conformations. Hybrid molecular mechanics/quantum mechanics calculations show <1.2 kcal/mol strain for any of the small-molecule structures. Extrapolation of the NaI cellobiose geometry to a cellulose molecule gives a left-handed helix with 2.9 residues per turn. The energy map and small-molecule crystal structures imply that cellulose helices having 2.5 and 3.0 residues per turn are left-handed.     

Statistical and molecular dynamics studies of buried waters in globular proteins

Buried solvent molecules are common in the core of globular proteins and contribute to structural stability. Folding necessitates the burial of polar backbone atoms in the protein core, whose hydrogen-bonding capacities should be satisfied on average. Whereas the residues in alpha-helices and beta-sheets form systematic main-chain hydrogen bonds, the residues in turns, coils and loops often contain polar atoms that fail to form intramolecular hydrogen bonds. The statistical analysis of 842 high resolution protein structures shows that well-resolved, internal water molecules preferentially reside near residues without alpha-helical and beta-sheet secondary structures. These buried waters most often form primary hydrogen bonds to main-chain atoms not involved in intramolecular hydrogen bonds, providing strong evidence that hydrating main-chain atoms is a key structural role of buried water molecules. Additionally, the average B-factor of protein atoms hydrogen-bonded to waters is smaller than that of protein atoms forming intramolecular hydrogen bonds, and the average B-factor of water molecules involved in primary hydrogen bonds with main-chain atoms is smaller than the average B-factor of water molecules involved in secondary hydrogen bonds to protein atoms that form concurrent intramolecular hydrogen bonds. To study the structural coupling between internal waters and buried polar atoms in detail we simulated the dynamics of wild-type FKBP12, in which a buried water, Wat137, forms one side-chain and multiple main-chain hydrogen bonds. We mutated E60, whose side-chain hydrogen bonds with Wat137, to Q, N, S or A, to modulate the multiplicity and geometry of hydrogen bonds to the water. Mutating E60 to a residue that is unable to form a hydrogen bond with Wat137 results in reorientation of the water molecule and leads to a structural readjustment of residues that are both near and distant to the water. We predict that the E60A mutation will result in a significantly reduced affinity of FKBP12 for its ligand FK506. The propensity of internal waters to hydrogen bond to buried polar atoms suggests that ordered water molecules may constitute fundamental structural components of proteins, particularly in regions where alpha-helical or beta-sheet secondary structure is not present.     

Structure of papain refined at 1.65 A resolution

Papain is a sulfhydryl protease from the latex of the papaya fruit. Its molecules consist of one polypeptide chain with 212 amino acid residues. The chain is folded into two domains with the active site in a groove between the domains. We have refined the crystal structure of papain, in which the sulfhydryl group was oxidized, by a restrained least-squares procedure at 1.65 A to an R-factor of 16.1%. The estimated accuracy in the atomic co-ordinates is 0.1 A, except for disordered atoms. All phi/psi angles for non-glycine residues are found within the outer limit boundary of a Ramachandran plot and this provides another check on the quality of the model. In the alpha-helical parts of the structure, the C = O bonds are directed more away from the helix axis than in a classical alpha-helix, leading to somewhat longer hydrogen bonds, 2.98 A, compared to 2.89 A. The hydrogen-bonding parameters and conformational angles in the anti-parallel beta-sheet structure show a large diversity. Hydrogen bonds in the core of the sheet are generally shorter than those at the more twisted ends. The average value is 2.91 A. The hydrogen bond distance Ni+3-Oi in turns is relatively long and the geometry is far from linear. Hydrogen bond formation, therefore, is perhaps not an essential prerequisite for turn formation. Although the crystallization medium is 62% (w/w) methanol in water, only 29 out of 224 solvent molecules can be regarded with any certainty as methanol molecules. The water molecules play an important role in maintaining structural stability. This is specially true for internal water. Twenty-one water molecules are located in contact areas between adjacent papain molecules. It seems as if the enzyme is trapped in a grid of water molecules with only a limited number of direct interactions between the protein molecules. The residues in the active site cleft belong to the most static parts of the structure. In general, disorder in atomic positions increases when going from the interior of the protein molecule to its surface. This behavior was quantified and it was found that the point of minimum disorder is near the molecular centroid.     

Molecular conformation of achatin-I, an endogenous neuropeptide containing D-amino acid residue. X-ray crystal structure of its neutral form

The molecular conformation of achatin-I neutral form (H-Gly-D-Phe-Ala-Asp-OH), an endogenous neuropeptide, was elucidated by X-ray crystal analysis. The molecule has a type II' beta-turn structure with the D-Phe-Ala residues at the corner of the bend, which is further stabilized by two NH(Gly)...C gamma = O sigma(Asp) and NH(Asp)...C gamma = O sigma(Asp) intramolecular hydrogen bonds. This turn conformation may be an important feature of achatin-I related to its neuroexcitatory activity.     

Structure and stability of the P93G variant of ribonuclease A.

The peptide bonds preceding Pro 93 and Pro 114 of bovine pancreatic ribonuclease A (RNase A) are in the cis conformation. The trans-to-cis isomerization of these bonds had been indicted as the slow step during protein folding. Here, site-directed mutagenesis was used to replace Pro 93 or Pro 114 with a glycine residue, and the crystalline structure of the P93G variant was determined by X-ray diffraction analysis to a resolution of 1.7 A. This structure is essentially identical to that of the wild-type protein, except for the 91-94 beta-turn containing the substitution. In the wild-type protein, the beta-turn is of type VIa. In the P93G variant, this turn is of type II with the peptide bond preceding Gly 93 being trans. The thermal stabilities of the P93G and P114G variants were assessed by differential scanning calorimetry and thermal denaturation experiments monitored by ultraviolet spectroscopy. The value of delta deltaGm which reports on the stability lost in the variants, is 1.5-fold greater for the P114G variant than for the P93G variant. The greater stability of the P93G variant is likely due to the relatively facile accommodation of residues 91-94 in a type II turn, which has a preference for a glycine residue in its i + 2 position.     

Unique side chain conformation of a Leu residue in a triple-helical structure

Single crystal structures of host-guest peptides, (Pro-Hyp-Gly)(4)-Leu-Hyp-Gly-(Pro-Hyp-Gly)(5) (LOG1) and (Pro-Hyp-Gly)(4)- (Leu-Hyp-Gly)(2)-(Pro-Hyp-Gly)(4) (LOG2), have been determined at 1.6 A and 1.4 A resolution, respectively. In these crystals, the side chain conformations of the Leu residues were (+)gauche-trans. This conformational preference for the Leu side chain in the Leu-Hyp-Gly sequence was explained by stereochemical considerations together with statistical analysis of Protein Data Bank data. In the (+)gauche-trans conformation, the Leu side chain can protrude along the radial direction of the rod-like triple-helical molecule. One strong hydrophobic interaction of the Leu residue was observed between adjacent molecules in the LOG2 crystal. Because the Leu-Hyp-Gly sequence is one of the most frequently occurring triplets in Type I collagen, this strong hydrophobic interaction can be expected in a fibrillar structure of native collagen. All the Leu residues in the asymmetric unit of the LOG1 and LOG2 crystals had water molecules hydrogen bonded to their NH. These water molecules made three additional hydrogen bonds with the Hyp OH, the Gly O[double bond]C, and a water molecule in the second hydration shell, forming a tetrahedral coordination of hydrogen bonds, which allows a smaller mean-square displacement factor of this water oxygen atom than those of other water molecules. These hydrogen bonds stabilize the molecular and packing structures by forming one O[double bond]C(Gly)---W---OH(Hyp) intra-molecular linkage and two NH(Leu)---W---O[double bond]C(Gly) and NH(Leu)---W---OH(Hyp) inter-molecular linkages.     

The pseudomolecule method and the structure of globular proteins. II. The example of ribonuclease F1 and T1

The pseudomolecule approach to the structure of globular proteins in which a small number of water molecules are incorporated into the "molecule" is tested again by comparing the ribbon of hydrogen bonds in two proteins, ribonuclease F1 and T1. These two molecules are 59% homologous and have the same backbone conformation both globally and locally. The two ribbons of hydrogen bonds that cover the whole of the backbone are conserved with an accuracy of some 95% providing that allowance is made for the intrusion into one of the pair of such extra factors as the presence of adducts or metal ions, the insertions and the absence of a few water molecules from one of the x-ray data sets. Without these corrections, the conservation of the ribbon is some 85%. There are 35 conserved hydrogen-bonding residues, nearly all of which show many unions to the backbone or interactions with the active site. There are 36 point mutations that involve one or two hydrogen-bonding side chains and nearly all of these have either none or one hydrogen bond to the backbone. These are minor contributors to the ribbon of hydrogen bonds. Of the 71 residues involved in these two categories, all but six fit into the pseudomolecular picture of the structure of globular proteins. The remaining 30 residues almost all contain conserved hydrocarbon side chains that may have a second order effect on the structure through their space filling effects.     

Refined structure of human carbonic anhydrase II at 2.0 Å resolution

The structure of human erythrocytic carbonic anhydrase II has been refined by constrained and restrained structure–factor least-squares refinement at 2.0 Å resolution. The conventional crystallographic R value is 17.3%. Of 167 solvent molecules associated with the protein, four are buried and stabilize secondary structure elements. The zinc ion is ligated to three histidyl residues and one water molecule in a nearly tetrahedral geometry. In addition to the zinc-bound water, seven more water molecules are identified in the active site. Assuming that Glu-106 is deprotonated at pH 8.5, some of the hydrogen bond donor–acceptor relations in the active site can be assigned and are described here in detail. The Oγ1 atom of Thr-199 donates its proton to the Oε1 atom of Glu-106 and can function as a hydrogen bond acceptor only in additional hydrogen bonds.     

Four-stranded coiled-coil elastic protein in the byssus of the giant clam, Tridacna maxima

An elastic protein with a secondary structure distinct from all well-known load-bearing proteins is found in the byssus of the giant clam, Tridacna maxima . The byssus consists of a bundle of hundreds of individual threads, each measuring about about 100 μm in diameter, which exhibit a tendon-like mechanical response. The amino acid composition of Tridacna byssus, however, is unlike tendon collagen, lacking high glycine, proline, and hydroxyproline. Wide-angle X-ray scattering (WAXS) and small-angle X-ray scattering (SAXS) measurements suggest that the constituent nanofibrils of the byssal threads are distinct from known secondary structure motifs previously reported for elastic proteins including the collagen triple-helix, the β-sheet nanocrystalline domains of silks, or the double-stranded coiled-coil regions of intermediate filaments. Instead, X-ray diffraction data indicate a structural organization in which four coiled-coil α-helices form a stable rope-like structure, which then further pack in a pseudohexagonal lattice to form nanofibrils. Amino acid composition analysis shows unusually high concentrations of acidic as well as basic residues, suggesting that the four-helix structure is stabilized by strong ionic interactions between oppositely charged residues in neighboring strands. The composition also suggests additional stabilization by disulfide cross-linking. On a larger scale, scanning and conventional transmission electron microscope (STEM and TEM) observations indicate that the nanofibrils exhibit an alternating periodicity of about 500 nm along the axial direction. A molecular model that combines the mechanical properties with the structural characteristics of the Tridacna byssal threads is proposed.     

A view of the three dimensional structure of globular proteins

A view of the three dimensional structure of globular proteins based on continuous networks of hydrogen bonds is proposed. Active sites of enzymes and ion sites are prominent and, within the networks, there are islands of hydrophobic regions giving an overall piebald effect to the appearance of the molecule. This point of view was originally suggested by the results of quantum mechanical computations on the coupling between hydrogen bonds. A formalism for the total energy of a globular protein in water is also suggested.The study of five lines of experimental evidence supports this suggestion. The analysis of the experimental X-ray data for ten globular proteins, using the NETWORK program, revealed the existence of these hydrogen bond networks; X-ray data showed that water molecules tend to occupy fixed positions relative to the protein molecule; a survey has shown that water molecules tend to occupy specific positions relative to the hydrogen bonding side chains; experimental evidence on the bulk properties of lysozyme showed that there exist tightly bound water molecules; graphics studies of the ribonucleaseA molecule demonstrated the networks and the piebald effect. This point of view is pictorially simple and, to illustrate the use of such networks, we discuss the simple ion pairs which occur as substructures within the networks.     

Helical structure of β(1–3) xylan and the water mediated hydrogen bonding schemes

A six-fold intertwined triple helical structure for the polysaccharide β(1–3) xylan was generated with the axial advance of 0·306 nm per residue. A stereochemically possible site for the water molecule has been determined and water mediated intrachain and interchain hydrogen bond schemes are possible for the right-handed triple helical structure, whereas only interchain hydrogen bonding appears plausible in the left-handed triple helical structure. The water mediated hydrogen bond is almost linear. X-ray refinement using a Linked-Atom Least-Squares (LALS) procedure has enabled us to determine the orientation of the molecule in the hexagonal unit cell, locate the position of the water molecules and yield a reliability index, R, of 0·35. The refined model in this present study confirms the original chirality of an earlier model but differs in the water mediated hydrogen bonding scheme.