Structure of the type I collagen molecule based on conformational energy computations: the triple-stranded helix and the N-terminal telopeptide.

Various studies have implicated a crucial role for the non-helical ends (telopeptides) of the collagen molecule during fibrillogenesis. In this paper, the first extensive conformational analysis of the type I collagen N-terminal telopeptide is reported. The commonly used "build-up" procedure for peptides and proteins has been used, with relevant modifications to take account of all the stereochemical constraints affecting the telopeptide. In particular, consideration was given not only to the interactions among the three chains that constitute the telopeptide, but also to the interactions between the telopeptide and the covalently linked triple helix. The computations led to a limited number of different structures within an energy range of 25 kcal/mol. Comparison of these models clearly shows that the portion of the telopeptide linked to the triple helix is rather rigid whereas its N terminus is more flexible. Furthermore, the lowest-energy structure has an energy that is markedly lower (by 7.75 kcal/mol) than that of other conformations with different structural features. The lowest-energy model of the N-terminal telopeptide, which differs from previous proposed models, has a contracted conformation compared to the triple helix region, in agreement with X-ray and neutron diffraction data on collagen fibers. Finally, the side-chains of the lysine residues of the telopeptide, involved in intermolecular cross-links in mature collagen fibers, are oriented to protrude to the exterior, in positions to interact with adjacent collagen molecules.

Crystallization of a hyperthermophilic archaeal elongation factor 1 alpha.

Intact and fully active elongation factor aEF-1 alpha from the hyperthermophilic archaeon Sulfolobus solfataricus has been crystallized as a complex with GDP. Crystals were stable at temperatures below 8 degrees C and showed significant diffraction beyond 3.0 A. The orthorhombic lattice parameters were a = 62.9 A, b = 81.3 A, c = 115.6 A with one molecule per asymmetric unit.

The ultrahigh resolution crystal structure of ribonuclease A containing an isoaspartyl residue: hydration and sterochemical analysis.

Crystals of the deamidated form of bovine pancreatic ribonuclease which contains an isoaspartyl residue in position 67 diffract to 0. 87 A at 100 K. We have refined the crystallographic model using anisotropic displacement parameters for all atoms to a conventional crystallographic residual R=0.101 for all observed reflections in the resolution range 61.0-0.87 A. The ratio observations/parameters is 7.2 for the final model. This structure represents one of the highest resolution protein structures to date and interestingly, it is the only example containing more than one molecule in the asymmetric unit with a resolution better than 1.0 A. The non-crystallographic symmetry has been used as a validation check of the geometrical parameters and it has allowed an estimate for an upper limit of errors associated with this high resolution model. In the present structure it was possible to obtain a more accurate picture of the active site whose electron density was not clearly interpretable in the previous 1.9 A resolution structure. In particular, the P1 site is alternatively occupied either by a sulphate anion or by a water molecule network. Most of hydrogen atoms were visible in the electron density maps, including those involved in C(alpha)-H(alpha).O interactions. Analysis of protein-solvent interactions has revealed the occurrence of an extensive cluster of water molecules, predominantly arranged in pentagonal fused rings and surrounding hydrophobic moiety of side-chains. Finally, in spite of the limited sample of residues, we have detected a clear dependence of backbone N-C(alpha)-C angle on residue conformation. This correlation can be fruitfully used as a valuable tool in protein structure validation.

Protein titration in the crystal state.

Proteins are complex structures whose overall stability critically depends on a delicate balance of numerous interactions of similar strength, which are markedly influenced by their environment. Here, we present an analysis of the effect of pH on a protein structure in the crystalline state using RNase A as a model system. By altering only one physico-chemical parameter in a controlled manner, we are able to quantify the structural changes induced in the protein. Atomic resolution X-ray diffraction data were collected for crystals at six pH* values ranging from 5.2 to 8.8, and the six independently refined structures reveal subtle, albeit well-defined variations directly related to the pH titration of the protein. The deprotonation of the catalytic His12 residue is clearly evident in the electron density maps, confirming the reaction mechanism proposed by earlier enzymatic and structural studies. The concerted structural changes observed in the regions remote from the active-site point to an adaptation of the protein structure to the changes in the physico-chemical environment. Analysis of the stereochemistry of the six structures provided accurate estimates of p Kavalues of most of the histidine residues. This study gives further evidence for the advantage of atomic resolution X-ray crystallographic analyses for revealing small but significant structural changes which provide clues to the function of a biological macromolecule.

Crystal structure of Trematomus newnesi haemoglobin re-opens the root effect question.

As new structural data have become available, somewhat contrasting explanations of the Root effect in fish haemoglobins (Hb) have been provided. Hb 1 of the Antarctic fish Trematomus newnesi has a nearly pH-independent oxygen affinity, in spite of 95 % sequence identity with Hb 1 of Trematomus (previously named Pagothenia) bernacchii that has a strong Root effect. Here, the 2.2 A R-state structure of Trematomus newnesi Hb 1 is presented. The structure is similar to that of Root effect fish Hbs from Spot and T. bernacchii, suggesting that the differences in the pH dependence cannot be related to the modulation of the R-state. In comparison to T. bernacchii Hb 1, the role of the three mutations Thr41 (C6)alpha-->Ile, Ala97 (G3)alpha-->Ser and His41 (C7)beta-->Tyr at the alpha1beta2-interface is discussed.

Deamidation in proteins: the crystal structure of bovine pancreatic ribonuclease with an isoaspartyl residue at position 67.

The non-enzymatic deamidation of asparagine residues in proteins is a widely occurring reaction, both in vivo and in vitro. Although the importance of this process is commonly recognised, only little structural information is available on it. In order to evaluate the structural effects of this reaction in proteins, we have determined the crystal structure of a ribonuclease A derivative in which asparagine 67 has been replaced by an isoaspartyl residue, as a consequence of an in vitro deamidation reaction. The overall structure of the model, refined to a crystallographic R-factor of 0.159 at a resolution of 1.9 A, is very similar to that of the native protein, but considerable deviations are observed in the region delimited by the disulphide bridge 65-72. In particular, the insertion of an extra methylene group in the main chain at residue 67 breaks up the hydrogen bond network that makes this region rather rigid in ribonuclease A. On the basis of the structure observed, some of the slightly but significantly different properties of this deamidated derivative, with respect to the native enzyme, can be explained.

A potential allosteric subsite generated by domain swapping in bovine seminal ribonuclease.

Bovine seminal ribonuclease (BS-RNase) is a peculiar member of the pancreatic-like ribonuclease superfamily endowed with unique biological functions. It has been shown that native BS-RNase is a mixture of two distinct dimeric forms. The most abundant form is characterised by the swapping of the N-terminal helix. Kinetic studies have shown that this dimer is allosterically regulated, whereas the minor component, in which no swapping occurs, exhibits typical Michaelian kinetics. In order to correlate the catalytic properties with the structural features of BS-RNase, we have determined the crystal structure of the BS-RNase swapping dimer complexed with uridylyl(2'-5')guanosine. The structure of the complex was refined to an R value of 0.189 at 1.9 A resolution. Surprisingly, the enzyme binds four dinucleotide molecules, all in a non-productive way. In the two active sites, the guanine base is located in the subsite that is specific for pyrimidines. This unusual binding has been observed also in complexes of RNase A with guanine-containing nucleotides (retro-binding). One of the two additional dinucleotide molecules bound to the enzyme is located on the surface of the protein in a pocket generated by crystal packing; the second was found in a cavity at the interface between the two subunits of the swapping dimer. There are indications that the interface site plays a role in the allosteric regulation exhibited by BS-RNase. This finding suggests that domain swapping may not merely be a mechanism that proteins adopt for the transition from a monomeric to oligomeric state but can be used to achieve modulations in catalytic function.

X-ray crystallographic determination of a collagen-like peptide with the repeating sequence (Pro-Pro-Gly).

The crystal structure of the triple-helical peptide (Pro-Pro-Gly)10 has been re-determined to obtain a more accurate description for this widely studied collagen model and to provide a comparison with the recent high-resolution crystal structure of a collagen-like peptide containing Pro-Hyp-Gly regions. This structure demonstrated that hydroxyproline participates extensively in a repetitive hydrogen-bonded assembly between the peptide and the solvent molecules. Two separate structural studies of the peptide (Pro-Pro-Gly)10 were performed with different crystallization conditions, data collection temperatures, and X-ray sources. The polymer-like structure of one triple-helical repeat of Pro-Pro-Gly has been determined to 2.0 A resolution in one case and 1.7 A resolution in the other. The solvent structures of the two peptides were independently determined specifically for validation purposes. The two structures display a reverse chain trace compared with the original structure determination. In comparison with the Hyp-containing peptide, the two Pro-Pro-Gly structures demonstrate very similar molecular conformation and analogous hydration patterns involving carbonyl groups, but have different crystal packing. This difference in crystal packing indicates that the involvement of hydroxyproline in an extended hydration network is critical for the lateral assembly and supermolecular structure of collagen.

Productive and nonproductive binding to ribonuclease A: X-ray structure of two complexes with uridylyl(2',5')guanosine.

Guanine-containing mono- and dinucleotides bind to the active site of ribonuclease A in a nonproductive mode (retro-binding) (Aguilar CF, Thomas PJ, Mills A, Moss DS, Palmer RA. 1992. J Mol Biol 224:265-267). Guanine binds to the highly specific pyrimidine site by forming hydrogen bonds with Thr45 and with the sulfate anion located in the P1 site. To investigate the influence of the anion present in the P1 site on retro-binding, we determined the structure of two new complexes of RNase A with uridylyl(2',5')guanosine obtained by soaking two different forms of pre-grown RNase A crystals. In one case, RNase A was crystallized without removing the sulfate anion strongly bound to the active site; in the other, the protein was first equilibrated with a basic solution to displace the anion from the P1 site. The X-ray structures of the complexes with and without sulfate in P1 were refined using diffraction data up to 1.8 A (R-factor 0.192) and 2.0 A (R-factor 0.178), respectively. The binding mode of the substrate analogue to the protein differs markedly in the two complexes. When the sulfate is located in P1, we observe retro-binding; whereas when the anion is removed from the active site, the uridine is productively bound at the B1 site. In the productive complex, the electron density is very well defined for the uridine moiety, whereas the downstream guanine is disordered. This finding indicates that the interactions of guanine in the B2 site are rather weak and that this site is essentially adenine preferring. In this crystal form, there are two molecules per asymmetric unit, and due to crystal packing, only the active site of one molecule is accessible to the ligand. Thus, in the same crystal we have a ligand-bound and a ligand-free RNase A molecule. The comparison of these two structures furnishes a detailed and reliable picture of the structural alterations induced by the binding of the substrate. These results provide structural information to support the hypotheses on the role of RNase A active site residues that have recently emerged from site-directed mutagenesis studies.

Pyramidalization of backbone carbonyl carbon atoms in proteins.

The high accuracy of X-ray analyses at atomic resolution is now able to display subtle deformations from standard geometry of building blocks in proteins. From the analysis of nine ultra-high resolution protein structures, we derived the first experimental evidence that a significant pyramidalization at the main-chain carbonyl carbon atom occurs in proteins. Our findings also show that this pyramidalization is related to the main-chain psi torsion angle. The carbonyl carbon atoms of residues that adopt alphaR and extended conformations show a clear preference for positive and negative pyramidalization, respectively. The agreement between our data and those previously obtained from small molecule structures demonstrates that carbon pyramidalization is an intrinsic property of the peptide structure. Although small in magnitude, the pyramidalization is well preserved in the complex folded state of a macromolecular structure that results from the interplay of many different forces. In addition, this property of the peptide group may have interesting implications for the enzymatic reactions involving the carbonyl carbon atoms.

Preferred proline puckerings in cis and trans peptide groups: implications for collagen stability.

The interplay between side-chain and main-chain conformations is a distinctive characteristic of proline residues. Here we report the results of a statistical analysis of proline conformations using a large protein database. In particular, we found that proline residues with the preceding peptide bond in the cis state preferentially adopt a down puckering. Indeed, out of 178 cis proline residues, as many as 145 (81%) are down. By analyzing the 1-4 and 1-5 nonbonding distances between backbone atoms, we provide a structural explanation for the observed trend. The observed correlation between proline puckering and peptide bond conformation suggests a new mechanism to explain the reported shift of the cis-trans equilibrium in proline derivatives. The implications of these results for the current models of collagen stability are also discussed.

Binding of a substrate analog to a domain swapping protein: X-ray structure of the complex of bovine seminal ribonuclease with uridylyl(2',5')adenosine.

Bovine seminal ribonuclease (BS-RNase) is a unique member of the pancreatic-like ribonuclease superfamily. The native enzyme is a mixture of two dimeric forms with distinct structural features. The most abundant form is characterized by the swapping of N-terminal fragments. In this paper, the crystal structure of the complex between the swapping dimer and uridylyl(2',5')adenosine is reported at 2.06 A resolution. The refined model has a crystallographic R-factor of 0.184 and good stereochemistry. The quality of the electron density maps enables the structure of both the inhibitor and active site residues to be unambiguously determined. The overall architecture of the active site is similar to that of RNase A. The dinucleotide adopts an extended conformation with the pyrimidine and purine base interacting with Thr45 and Asn71, respectively. Several residues (Gln11, His12, Lys41, His119, and Phe120) bind the oxygens of the phosphate group. The structural similarity of the active sites of BS-RNase and RNase A includes some specific water molecules believed to be relevant to catalytic activity. Upon binding of the dinucleotide, small but significant modifications of the tertiary and quaternary structure of the protein are observed. The ensuing correlation of these modifications with the catalytic activity of the enzyme is discussed.

Novel six-nucleotide deletion in the hepatic fructose-1,6-bisphosphate aldolase gene in a patient with hereditary fructose intolerance and enzyme structure-function implications.

Hereditary fructose intolerance (HFI) is an autosomal recessive human disease that results from the deficiency of the hepatic aldolase isoenzyme. Affected individuals will succumb to the disease unless it is readily diagnosed and fructose eliminated from the diet. Simple and non-invasive diagnosis is now possible by direct DNA analysis that scans for known and unknown mutations. Using a combination of several PCR-based methods (restriction enzyme digestion, allele specific oligonucleotide hybridisation, single strand conformation analysis and direct sequencing) we identified a novel six-nucleotide deletion in exon 6 of the aldolase B gene (delta 6ex6) that leads to the elimination of two amino acid residues (Leu182 and Val183) leaving the message inframe. The three-dimensional structural alterations induced in the enzyme by delta 6ex6 have been elucidated by molecular graphics analysis using the crystal structure of the rabbit muscle aldolase as reference model. These studies showed that the elimination of Leu182 and Val183 perturbs the correct orientation of adjacent catalytic residues such as Lys146 and Glu187.

Crystal structure of the dimeric unswapped form of bovine seminal ribonuclease.

Bovine seminal ribonuclease is a unique case of protein dimorphism, since it exists in two dimeric forms, with different biological and kinetic behavior, which interconvert into one another through three-dimensional swapping. Here we report the crystal structure, at 2.2 A resolution, of the unswapped form of bovine seminal ribonuclease. Besides completing the structural definition of bovine seminal ribonuclease conformational dimorphism, this study provides the structural basis to explain the dependence of the enzyme cooperative effects on its swapping state.

Identification of aminosuccinyl residues in peptides by second-derivative ultraviolet spectrometry.

A second derivative ultraviolet spectrophotometric method for direct identification of aminosuccinyl peptides is described. The advantages and limits of this procedure have been assessed on the basis of results of five model peptides including the aminosuccinyl moiety.

Spontaneous splenic vein aneurysm rupture in the puerperium.

A case of intra- and retroperitoneal haemorrhage after a splenic vein aneurysm rupture is described. A 27-year-old woman complained of chest pain on her first postpartum day. Symptoms were initially suggestive of pulmonary embolism, but within 3 h she developed severe acute abdominal pain, abdominal distension and haemodynamic instability. Ultrasound demonstrated a non-echogenic mass in the abdomen suggestive of a fluid collection and a computed tomography scan confirmed the presence of a lesion at the pancreatic tail. Urgent laparotomy revealed splenic vein rupture near the pancreatic tail. Partial pancreatectomy and splenectomy were performed. The patient subsequently made an uneventful recovery. Histological examination revealed a splenic vein aneurysm and chronic inflammatory changes in the pancreatic tissue. Rupture of a splenic vein aneurysm is a rare event and the diagnosis may present difficulty because its presentation is similar to several other more common conditions. However, rupture of a splenic vein aneurysm should be suspected in any pregnant woman with unexplained abdominal pain or with clear signs of haemorrhage, as delay in diagnosis can have devastating consequences.

Swapping structural determinants of ribonucleases: an energetic analysis of the hinge peptide 16-22.

Bovine seminal ribonuclease (BS-RNase) is a homodimeric enzyme strictly homologous to the pancreatic ribonuclease (RNase A). Native BS-RNase is an equilibrium mixture of two distinct dimers differing in the interchange of the N-terminal segments and in their biological properties. The loop 16-22 plays a fundamental role on the relative stability of the two isomers. Both the primary and tertiary structures of the RNase A differ substantially from those of the seminal ribonuclease in the loop region 16-22. To analyze the possible stable conformations of this loop in both enzymes, structure predictions have been attempted, according to a procedure described by Palmer and Scheraga [Palmer, K. A. & Scheraga, H. A. (1992) J. Comput. Chem. 13, 329-350]. Results compare well with experimental x-ray structures and clarify the structural determinants that are responsible for the swapping of the N-terminal domains and for the peculiar properties of BS-RNase. Minimal modifications of RNase A sequence needed to form a stable swapped dimer are also predicted.

Folding of aminosuccinyl peptides: thermodynamic data from temperature dependent circular dichroism measurements.

The conformational equilibrium of aminosuccinyl peptides between extended conformations and an intramolecularly hydrogen bonded type II' beta-turn conformation has been studied on the peptide Boc-L-Asu-Gly-L-Ala-OMe (Asu = aminosuccinyl residue) by means of temperature dependence of circular dichroism spectra. Owing to the peculiar chiroptical and conformational properties of the Asu residue, this technique proved to be very useful for deriving thermodynamic data for the above folding process. The value of delta H0 (-6.6 kJ mol-1), obtained for the peptide studied in a chloroformacetonitrile mixture, shows that the lower energy of the folded conformer is primarily due to the characteristic intramolecular hydrogen bond of the beta turns.

Deamidation via cyclic imide of asparaginyl peptides: dependence on salts, buffers and organic solvents.

The deamidation reaction of Asn side chain was studied in the presence of the chemicals usually used at high concentration in the purification and crystallization of peptides and proteins. All the experiments were performed on the model peptide Boc-L-Asn-Gly-Gly-NH2. The pathway of the reaction is not affected by the medium used; in all cases it proceeds through a succinimide intermediate giving a mixture of isoaspartyl and aspartyl peptide. However, the rate of the reaction significantly depends on the solvent: the addition of organic solvents to an aqueous solution of the peptide has the general effect of decreasing the reaction rate, which, on the other hand, is strongly enhanced by a high concentration of organic and inorganic buffers. Only a minor influence is exerted by aprotic salts and polyethylene glycol.