Sequence regularities and packing of collagen molecules

Fourier analysis of sequences along edges of the type I collagen molecule constructed from two α1(I) and one α2 chains shows that the molecule is two-sided if the supercoil pitch of the α chains along the molecular axis, P, is 39 residues ($\text{D}\text{6}$, where D = 234 residues or 67 nm). One side has alternating charged and hydrophobic regions with spacings of $\text{D}\text{6}$, while the other side has an excess of hydrophobic residues with a spacing of $\text{D}\text{11}$. These characteristics arise from sequence regularities in the α chains and the geometric relationship between the chains. The pattern is marginally strongest with α2 as chain 1. The $\text{D}\text{6}$ sides could form the inside of a helical microfibril where contacts between molecules would fall P apart along the α chains. The $\text{D}\text{11}$ sides could form the outside of the microfibril where contacts between microfibrils would be spaced apart by the α chain supercoil along the microfibril axis, P′. If the microfibril is a 5₄ helix of D-staggered collagen molecules with a left-handed supercoil of pitch $\text{20D}\text{11}$, P′ is close to $\text{2D}\text{11}$ (43 residues). $\text{2D}\text{11}$ subsets in the α chains give rise to the $\text{D}\text{11}$ spacing along the molecule. The microfibril has 4₁ screw symmetry satisfying X-ray diffraction evidence that microfibrils pack in a tetragonal unit cell.This model is the same as proposed previously by us (Trus & Piez, 1976: Piez & Trus, 1977) except that P = 39 rather than 30 residues. Contrary to our earlier assumption, P = 39 residues is within the range allowed by X-ray diffraction measurements. The present results favor P = 39 since it relates regularities in the α chain sequences to helical parameters in a direct way. Furthermore, model studies show that geometric arguments which support P = 30 are equally strong at P = 39 residues.     

A study of the sulfhydryl groups of rat brain hexokinase

Rat brain hexokinase (ATP: d-hexose-6-phosphotransferase, EC 2.7.1.1) is rapidly inactivated by reaction with 5,5′-dithiobis-(2-nitrobenzoate). The inactivation follows monophasic first-order kinetics in either the absence of ligands (k = 0.641 min^?1 at 25 °C) or in the presence of saturating levels of ATP (free or complexed with Mg²⁺) or P₁; the inactivation rate is slightly increased ($\text{k ? 0.7}$ min ^?1) in the presence of ATP or P₁. In contrast, glucose and glucose-6-P markedly decrease the inactivation rate; inactivation in the presence of these ligands is biphasic, with two first-order rates ($\text{k ? 0.5}$ min^?1 and 0.01 min^?1) being distinguishable.The enzyme contains 14 sulfhydryl groups which react with 5,5′-dithiobis-(2-nitrobenzoate); reaction of these groups in the native enzyme is complete after 2 hr at 25 °C, or in approx 5 min with the urea or guanidine-denatured enzyme. In the native enzyme, three classes of sulfhydryl groups are distinguishable and are designated as F-, I-, or S-type based on their fast (k ? 0.7 min^?1), intermediate ($\text{k ? 0.5-0.7}$ min^?1), or slow ($\text{k ? 0.02}$ min^?1 rates of reaction with 5,5′-dithiobis-(2-nitrobenzoate). The correlation of inactivation rates with the rates for reaction of the I-type sulfhydryls indicates that the I-type sulfhydryls include residues necessary for catalytic activity. The F-type residues are clearly not required for activity.The effects of ATP, P₁, glucose, and glucose-6-P on the reactivity of the sulfhydryls have been determined. As in the absence of ligands, S-, I-, and F-type sulfhydryls could be distinguished. In the presence of saturating concentrations of these ligands, the F, I, and S classes of sulfhydryls contained respectively: with ATP, 1, 4, and 7 residues; with P₁, 1, 3, and 7 residues; with glucose, 1, 2, and 5 residues; with glucose-6-P, 1, 2, and 1 residues. Comparison with rate constants for inactivation in the presence of these ligands again indicated that I-type sulfhydryls were particularly important in maintenance of enzyme activity. The present results indicate considerable similarity between the reactivity of the sulfhydryl residues in rat brain hexokinase and the sulfhydryls of the bovine brain enzyme [V. D. Redkar and U. W. Kenkare (1972), J. Biol. Chem., 247, 7576–7584].     

Comparative specificity of microbial acid proteinases for synthetic peptides. II. Effect of secondary interaction

To investigate the effect of “secondary interaction” on hydrolysis by various acid proteinases from molds and yeasts, synthetic peptides

amino acid residues) were used as substrates. Pepsin was used for the comparative study. These peptides were split at the peptide bonds indicated by the arrows, permitting examination of the effect of residue X distant by two or three amino acid residues from the hydrolytic site in the peptides. According to the system of Schechter and Berger (Biochem. Biophys. Res. Commun. 27; 157, 1967), the amino acid residues in peptide substrates were numbered P₁, P₂, etc. toward the N-terminal direction from the site of hydrolysis, and P₁′, P₂′, etc. toward the C-terminal direction. The results indicated that hydrolysis by these microbial enzymes is affected by at least six amino acid residues (P₁-P₃ and P₁′-P₃′) in peptide substrates, as is seen with pepsin. Elongation of the peptide chain with suitable amino acid residues from P₁ to P₂ or P₃ and from P₁′ to P₂′ or P₃′ in peptide substrates resulted in much or less increase of hydrolysis depending upon the species of the enzyme producers.     

Visualization of drug--nucleic acid interactions at atomic resolution. V. Structure of two aminoacridine--dinucleoside monophosphate crystalline complexes, proflavine--5-iodocytidylyl (3'-5') guanosine and acridine orange--5-iodocytidylyl (3'-5') guanosine

Acridine orange and proflavine form complexes with the dinucleoside monophosphate, 5-iodocytidylyl(3′–5′)guanosine. The acridine orange-iodoCpG² crystals are monoclinic, space group P2₁, with unit cell dimensions $\text{a = 14.36}\text{A}\text{?}\text{, b = 19.64}\text{A}\text{?}\text{, c = 20.67}\text{A}\text{?}$, β = 102.5 °. The proflavine-iodoCpG crystals are monoclinic, space group C2, with unit cell dimensions $\text{a = 32.14}\text{A}\text{?}\text{, b = 22.23}\text{A}\text{?}\text{, c = 18.42}\text{A}\text{?}$, β = 123.3 °. Both structures have been solved to atomic resolution by Patterson and Fourier methods, and refined by full matrix least-squares.Acridine orange forms an intercalative structure with iodoCpG in much the same manner as ethidium, ellipticine and 3,5,6,8-tetramethyl-N-methyl phenanthrolinium (Jain et al., 1977, Jain et al., 1979), except that the acridine nucleus lies asymmetrically in the intercalation site. This asymmetric intercalation is accompanied by a sliding of base-pairs upon the acridine nucleus and is similar to that observed with the 9-aminoacridine-iodoCpG asymmetric intercalative binding mode described in the previous papers (Sakore et al., 1977, Sakore et al., 1979). Basepairs above and below the drug are separated by about 6.8 Å and are twisted about 10 °; this reflects the mixed sugar puckering pattern observed in the sugar-phospate chains: C3′ endo (3′–5′) C2′ endo (i.e. each cytidine residue has a C3′ endo sugar comformation, while each guanosine residue has a C2′ endo sugar conformation), alterations in glycosidic torsional angles and other small but significant conformational changes in the sugar-phosphate backbone.Proflavine, on the other hand, demonstrates symmetric intercalation with iodoCpG. Hydrogen bonds connect amino groups on proflavine with phosphate oxygen atoms on the dinucleotide. In contrast to the acridine orange structure, base-pairs above and below the intercalative proflavine molecule are twisted about 36 °. The altered magnitude of this angular twist reflects the sugar puckering pattern that is observed: C3′ endo (3′–5′) C3′ endo. Since proflavine is known to unwind DNA in much the same manner as ethidium and acridine orange (Waring, 1970), one cannot use the information from this model system to understand how proflavine binds to DNA (it is possible, for example, that hydrogen bonding observed between proflavine and iodoCpG alters the intercalative geometry in this model system).Instead, we propose a model for proflavine-DNA binding in which proflavine lies asymmetrically in the intercalation site (characterized by the C3′ endo (3′–5′) C2′ endo mixed sugar puckering pattern) and forms only one hydrogen bond to a neighboring phosphate oxygen atom. Our model for proflavine-DNA binding, therefore, is very similar to our acridine orange-DNA binding model. We will describe these models in detail in this paper.     

Visualization of drug--nucleic acid interactions at atomic resolution. VI. Structure of two drug--dinucleoside monophosphate crystalline complexes, ellipticine--5-iodocytidylyy (3'-5') guanosine and 3,5,6,8-tetramethyl-N-methyl phenanthrolinium--5-iodocytidylyl (3'-5') guanosine

Ellipticine and 3,5,6,8-tetramethyl-N-methyl phenanthrolinium form complexes with the dinucleoside monophosphate, 5-iodocytidylyl(3′–5′)guanosine. These crystals are isomorphous: ellipticine-iodoCpG² crystals are monoclinic, space group P2₁ with $\text{a = 13.88}\text{A}\text{?}\text{, b = 19.11}\text{A}\text{?}\text{, c = 21.42}\text{A}\text{?}$, β = 105.4; TMP-iodoCpG crystals are monoclinic, space group P2₁, with $\text{a = 13.99}\text{A}\text{?}\text{, b = 19.12}\text{A}\text{?}\text{, c = 21.31}\text{A}\text{?}$, β = 104.9 °. Both structures have been solved to atomic resolution by Patterson and Fourier methods, and refined by full matrix least-squares.The asymmetric unit in the ellipticine-iodoCpG structure contains two ellipticine molecules, two iodoCpG molecules, 20 water molecules and 2 methanol molecules, a total of 144 atoms, whereas, in the tetramethyl-N-methyl phenanthrolinium-iodoCpG complex, the asymmetric unit contains two TMP molecules, two iodoCpG molecules, 17 water molecules and 2 methanol molecules, a total of 141 atoms. In both structures, the two iodoCpG molecules are hydrogenbonded together by guanine-cytosine Watson-Crick base-pairing. Adjacent base-pairs within this paired iodoCpG structure are separated by about 6.7 Å; this separation results from intercalative binding by one ellipticine (or TMP) molecule and stacking by the other ellipticine (or TMP) molecule above or below the base-pairs. Base-pairs within the paired nucleotide units are related by a twist of 10 to 12 °. The magnitude of this angular twist is related to conformational changes in the sugar-phosphate chains that accompany drug intercalation. These changes partly reflect the mixed sugar puckering pattern observed: C3′ endo (3′–5′) C2′ endo (i.e. both iodocytidine residues have C3′ endo conformations, whereas both guanosine residues have C2′ endo conformations), and additional small but systematic changes in torsional angles that involve the phosphodiester linkages and the C4′C5′ bond.The stereochemistry observed in these model drug-nucleic acid intercalative complexes is almost identical to that observed in the ethidium-iodoUpA and -iodoCpG complexes determined previously (Tsai et al., 1975a,b,1977; Jain et al., 1977). This stereochemistry is also very similar to that observed in the 9-aminoacridine-iodoCpG and acridine orange-iodoCpG complexes described in the preceding papers (Sakore et al., 1979 Reddy et al., 1979). We have already proposed this stereochemistry to provide a unified understanding of a large number of intercalative drug-DNA (and RNA) interactions (Sobell et al., 1977a,b), and discuss this aspect of our work further in this paper.     

The catalytic aspartate is protonated in the Michaelis complex formed between trypsin and an in vitro evolved substrate-like inhibitor: a refined mechanism of serine protease action

The mechanism of serine proteases prominently illustrates how charged amino acid residues and proton transfer events facilitate enzyme catalysis. Here we present an ultrahigh resolution (0.93 Å) x-ray structure of a complex formed between trypsin and a canonical inhibitor acting through a substrate-like mechanism. The electron density indicates the protonation state of all catalytic residues where the catalytic histidine is, as expected, in its neutral state prior to the acylation step by the catalytic serine. The carboxyl group of the catalytic aspartate displays an asymmetric electron density so that the O_δ2–C_γ bond appears to be a double bond, with O_δ2 involved in a hydrogen bond to His-57 and Ser-214. Only when Asp-102 is protonated on O_δ1 atom could a density functional theory simulation reproduce the observed electron density. The presence of a putative hydrogen atom is also confirmed by a residual mF_obs − DF_calc density above 2.5 σ next to O_δ1. As a possible functional role for the neutral aspartate in the active site, we propose that in the substrate-bound form, the neutral aspartate residue helps to keep the pK_a of the histidine sufficiently low, in the active neutral form. When the histidine receives a proton during the catalytic cycle, the aspartate becomes simultaneously negatively charged, providing additional stabilization for the protonated histidine and indirectly to the tetrahedral intermediate. This novel proposal unifies the seemingly conflicting experimental observations, which were previously seen as either supporting the charge relay mechanism or the neutral pK_a histidine theory.