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The structures of metalloproteins that use redox-active metals for catalysis are usually exquisitely folded in a way that they are prearranged to accept their metal cofactors. Peptidylglycine α-hydroxylating monooxygenase (PHM) is a dicopper enzyme that catalyzes hydroxylation of the α-carbon of glycine-extended peptides for the formation of des-glycine amidated peptides. Here, we present the structures of apo-PHM and of mutants of one of the copper sites (H107A, H108A, and H172A) determined in the presence and absence of citrate. Together, these structures show that the absence of one copper changes the conformational landscape of PHM. In one of these structures, a large interdomain rearrangement brings residues from both copper sites to coordinate a single copper (closed conformation) indicating that full copper occupancy is necessary for locking the catalytically competent conformation (open). These data suggest that in addition to their required participation in catalysis, the redox-active metals play an important structural role. Sweta Maheshwari et al. present X-ray crystal structures of the two-copper enzyme peptidylglycine α-hydroxylating monooxygenase and three inactive mutant forms. They show that full copper occupancy is needed to maintain the catalytically competent (open) conformation of the enzyme.

Mutations in oncogenes often promote tumorigenesis by changing the conformation of the encoded proteins, thereby altering enzymatic activity. The PIK3CA oncogene, which encodes p110α, the catalytic subunit of phosphatidylinositol 3-kinase alpha (PI3Kα), is one of the two most frequently mutated oncogenes in human cancers. We report the structure of the most common mutant of p110α in complex with two interacting domains of its regulatory partner (p85α), both free and bound to an inhibitor (wortmannin). The N-terminal SH2 (nSH2) domain of p85α is shown to form a scaffold for the entire enzyme complex, strategically positioned to communicate extrinsic signals from phosphopeptides to three distinct regions of p110α. Moreover, we found that Arg-1047 points toward the cell membrane, perpendicular to the orientation of His-1047 in the WT enzyme. Surprisingly, two loops of the kinase domain that contact the cell membrane shift conformation in the oncogenic mutant. Biochemical assays revealed that the enzymatic activity of the p110α His1047Arg mutant is differentially regulated by lipid membrane composition. These structural and biochemical data suggest a previously undescribed mechanism for mutational activation of a kinase that involves perturbation of its interaction with the cellular membrane.

Drug resistance is a major problem affecting the clinical efficacy of antiretroviral agents, including protease inhibitors, in the treatment of infection with human immunodeficiency virus type 1 (HIV-1)/AIDS. Consequently, the elucidation of the mechanisms by which HIV-1 protease inhibitors maintain antiviral activity in the presence of mutations is critical to the development of superior inhibitors. Tipranavir, a nonpeptidic HIV-1 protease inhibitor, has been recently approved for the treatment of HIV infection. Tipranavir inhibits wild-type protease with high potency ( K i = 19 pM) and demonstrates durable efficacy in the treatment of patients infected with HIV-1 strains containing multiple common mutations associated with resistance. The high potency of tipranavir results from a very large favorable entropy change (− T Δ S = −14.6 kcal/mol) combined with a favorable, albeit small, enthalpy change (Δ H = −0.7 kcal/mol, 25°C). Characterization of tipranavir binding to wild-type protease, active site mutants I50V and V82F/I84V, the multidrug-resistant mutant L10I/L33I/M46I/I54V/L63I/V82A/I84V/L90M, and the tipranavir in vitro-selected mutant I13V/V32L/L33F/K45I/V82L/I84V was performed by isothermal titration calorimetry and crystallography. Thermodynamically, the good response of tipranavir arises from a unique behavior: it compensates for entropic losses by actual enthalpic gains or by sustaining minimal enthalpic losses when facing the mutants. The net result is a small loss in binding affinity. Structurally, tipranavir establishes a very strong hydrogen bond network with invariant regions of the protease, which is maintained with the mutants, including catalytic Asp25 and the backbone of Asp29, Asp30, Gly48 and Ile50. Moreover, tipranavir forms hydrogen bonds directly to Ile50, while all other inhibitors do so by being mediated by a water molecule.

GDP-mannose mannosyl hydrolase (GDPMH) from E. coli catalyzes the hydrolysis of GDP-α- d -sugars to GDP and β- d -sugars by nucleophilic substitution with inversion at the anomeric C1 of the sugar, with general base catalysis by His-124. The 1.3 A X-ray structure of the GDPMH-Mg2+-GDP complex was used to model the complete substrate, GDP-mannose into the active site. The substrate is linked to the enzyme by 12 hydrogen bonds, as well as by the essential Mg2+. In addition, His-124 was found to participate in a hydrogen bonded triad: His-124-NδH⋯Tyr-127-OH⋯Pro-120(C O). The contributions of these hydrogen bonds to substrate binding and to catalysis were investigated by site-directed mutagenesis. The hydrogen bonded triad detected in the X-ray structure was found to contribute little to catalysis since the Y127F mutation of the central residue shows only 2-fold decreases in both kcat and Km. The GDP leaving group is activated by the essential Mg2+ which contributes at least 105-fold to kcat, and by nine hydrogen bonds, including those from Tyr-103, Arg-37, Arg-52, and Arg-65 (via an intervening water), each of which contribute factors to kcat ranging from 24- to 309-fold. Both Arg-37 and Tyr-103 bind the β-phosphate of the leaving GDP and are only 5.0 A apart. Accordingly, the R37Q/Y103F double mutant shows partially additive effects of the two single mutants on kcat, indicating cooperativity of Arg-37 and Tyr-103 in promoting catalysis. The extensive activation of the GDP leaving group suggests a mechanism with dissociative character with a cationic oxocarbenium-like transition state and a half-chair conformation of the sugar ring, as found with glycosidase enzymes. Accordingly, Asp-22 which contributes 102.1- to 102.6-fold to kcat, is positioned to both stabilize a developing cationic center at C1 and to accept a hydrogen bond from the C2–OH of the mannosyl group, and His-88, which contributes 102.3-fold to kcat, is positioned to accept a hydrogen bond from the C3–OH of the mannose facilitating its distortion to a half-chair conformation. Also, the fluorinated substrate GDP-2-fluoro-α- d -mannose, for which the oxocarbenium ion-like transition state centered at C1 would be destabilized by electron withdrawal, shows a 16-fold lower kcat and a 2.5-fold greater Km than does GDP-α- d -mannose. The product of the contributions to catalysis of Arg-37 and Tyr-103 (taking their cooperativity into account), Arg-52, Arg-65, Mg2+, Asp-22, His-124, and His-88 is ≥1019, which exceeds the 1012-fold rate acceleration produced by GDPMH by a factor ≥107. Hence, additional pairs or groups of catalytic residues must act cooperatively to promote catalysis.

Escherichia coli ADP-ribose (ADPR) pyrophosphatase (ADPRase), a Nudix enzyme, catalyzes the Mg(2+)-dependent hydrolysis of ADP-ribose to AMP and ribose 5-phosphate. ADPR hydrolysis experiments conducted in the presence of H(2)(18)O and analyzed by electrospray mass spectrometry showed that the ADPRase-catalyzed reaction takes place through nucleophilic attack at the adenosyl phosphate. The structure of ADPRase in complex with Mg(2+) and a nonhydrolyzable ADPR analogue, alpha,beta-methylene ADP-ribose, reveals an active site water molecule poised for nucleophilic attack on the adenosyl phosphate. This water molecule is activated by two magnesium ions, and its oxygen contacts the target phosphorus (P-O distance of 3.0 A) and forms an angle of 177 degrees with the scissile bond, suggesting an associative mechanism. A third Mg(2+) ion bridges the two phosphates and could stabilize the negative charge of the leaving group, ribose 5-phosphate. The structure of the ternary complex also shows that loop L9 moves fully 10 A from its position in the free enzyme, forming a tighter turn and bringing Glu 162 to its catalytic position. These observations indicate that as part of the catalytic mechanism, the ADPRase cycles between an open (free enzyme) and a closed (substrate-metal complex) conformation. This cycling may be important in preventing nonspecific hydrolysis of other nucleotides.

Regulation of cellular levels of ADP-ribose is important in preventing nonenzymatic ADP-ribosylation of proteins. The Escherichia coli ADP-ribose pyrophosphatase, a Nudix enzyme, catalyzes the hydrolysis of ADP-ribose to ribose-5-P and AMP, compounds that can be recycled as part of nucleotide metabolism. The structures of the apo enzyme, the active enzyme and the complex with ADP-ribose were determined to 1.9A, 2.7A and 2.3A, respectively. The structures reveal a symmetric homodimer with two equivalent catalytic sites, each formed by residues of both monomers, requiring dimerization through domain swapping for substrate recognition and catalytic activity. The structures also suggest a role for the residues conserved in each Nudix subfamily. The Nudix motif residues, folded as a loop-helix-loop tailored for pyrophosphate hydrolysis, compose the catalytic center; residues conferring substrate specificity occur in regions of the sequence removed from the Nudix motif. This segregation of catalytic and recognition roles provides versatility to the Nudix family.

Many bioactive peptides must be amidated at their carboxy terminus to exhibit full activity. Surprisingly, the amides are not generated by a transamidation reaction. Instead, the hormones are synthesized from glycine-extended intermediates that are transformed into active amidated hormones by oxidative cleavage of the glycine N-C alpha bond. In higher organisms, this reaction is catalyzed by a single bifunctional enzyme, peptidylglycine alpha-amidating monooxygenase (PAM). The PAM gene encodes one polypeptide with two enzymes that catalyze the two sequential reactions required for amidation. Peptidylglycine alpha-hydroxylating monooxygenase (PHM; EC 1.14.17.3) catalyzes the stereospecific hydroxylation of the glycine alpha-carbon of all the peptidylglycine substrates. The second enzyme, peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL; EC 4.3.2.5), generates alpha-amidated peptide product and glyoxylate. PHM contains two redox-active copper atoms that, after reduction by ascorbate, catalyze the reduction of molecular oxygen for the hydroxylation of glycine-extended substrates. The structure of the catalytic core of rat PHM at atomic resolution provides a framework for understanding the broad substrate specificity of PHM, identifying residues critical for PHM activity, and proposing mechanisms for the chemical and electron-transfer steps in catalysis. Since PHM is homologous in sequence and mechanism to dopamine beta-monooxygenase (DBM; EC 1.14.17.1), the enzyme that converts dopamine to norepinephrine during catecholamine biosynthesis, these structural and mechanistic insights are extended to DBM.

The most commonly quoted mechanism of the coupling between the electrochemical proton gradient and the formation of ATP from ADP and Pi assumes that all states of the F1 portion of the ATP synthase have β subunits in “tight,” “loose,” and “open” conformations. Models based on this assumption are inconsistent with some of the available experimental evidence. A mechanism that includes an additional β subunit conformation, “closed,” observed in the rat liver structure overcomes these difficulties.

Peptide amidation is a ubiquitous posttranslational modification of bioactive peptides. Peptidylglycine alpha-hydroxylating monooxygenase (PHM; EC 1.14.17.3), the enzyme that catalyzes the first step of this reaction, is composed of two domains, each of which binds one copper atom. The coppers are held 11 A apart on either side of a solvent-filled interdomain cleft, and the PHM reaction requires electron transfer between these sites. A plausible mechanism for electron transfer might involve interdomain motion to decrease the distance between the copper atoms. Our experiments show that PHM catalytic core (PHMcc) is enzymatically active in the crystal phase, where interdomain motion is not possible. Instead, structures of two states relevant to catalysis indicate that water, substrate and active site residues may provide an electron transfer pathway that exists only during the PHM catalytic cycle.

Lipoxygenases are enzymes involved in the metabolism of polyunsaturated fatty acids. One molecule of the complex lipoxygenase-3 (L3) contains one atom of iron per enzyme (approx, 95 kDa). The iron cofactor is neither coordinated by porphyrins (heme moiety, as in hemoglobin), nor surrounded by a cluster of sulfur atoms. The enzyme could be described as an elaborate coordination complex that serves as a catalyst in a typical redox process involving an iron cation wrapped in its “host” enzyme, molecular oxygen, and a substrate that undergoes peroxygenation to an organic peroxide. The structure of soybean lipoxygenase L3 has been solved and refined to a discrepancy factor R = 23%, for 2.6 A resolution data and 805 residues out of 857. The iron cation is buried inside the protein molecule. The active site shows three oxygen atoms and three nigrogen atoms as possible iron ligands in a pseudo- C 3v configuration, with nitrogen atoms grouped together at one side and oxygen atoms gathered at the opposite end of the pseudo-3-fold axis.

The three-dimensional structures of three variants of human β alcohol dehydrogenase have been determined to 2·5 A resolution. These three structures differ only in the amino acid at position 47 and the molecules occupying the alcohol binding site. Human β1 alcohol dehydrogenase has an Arg at position 47 and was crystallized in a complex with NAD(H) and cyclohexanol. A naturally occurring variant of β1 alcohol dehydrogenase, found in approximately 50% of the Asian population, possesses a His at position 47 (β2 or β47H) and was crystallized in a complex with NAD+ and the inhibitor 4-iodopyrazole. A site-directed mutant of β1 alcohol dehydrogenase in which a Gly is substituted for Arg47 (β47G) was crystallized in a complex with NAD+. By comparing both the common and unique features of these structures, it is clear that position 47 contributes significantly to the strength of protein-coenzyme interactions. The substitution of Arg47 by His produces an enzyme with a 100-fold lower affinity for coenzyme, but creates no larger changes in the enzyme structure. The substitution of Arg47 by Gly produces an enzyme with coenzyme binding characteristics more similar to the wild-type enzyme than to the enzyme with His at position 47, but the structure of the Gly47 variant exhibits differences in and around the coenzyme binding site. These changes involve a rigid-body rotation of the catalytic domain towards the coenzyme domain by approximately 0·8° and local rearrangements of amino acid side-chains, such as a 1·0 A movement of Lys228, relative to the β1 enzyme. These structural alterations may compensate for the loss of coenzyme interactions contributed by Arg47 and can explain the high affinity of the Gly47 variant for coenzyme.

In mammals, the hydroperoxidation of arachidonic acid by lipoxygenases leads to the formation of leukotrienes and lipoxins, compounds that mediate inflammatory responses. Lipoxygenases are dioxygenases that contain a nonheme iron and are present in many animal cells. Soybean lipoxygenase-1 is a single-chain, 839-residue protein closely related to mammalian lipoxygenases. The structure of soybean lipoxygenase-1 solved to 2.6 angstrom resolution shows that the enzyme has two domains: a 146-residue beta barrel and a 693-residue helical bundle. The iron atom is in the center of the larger domain and is coordinated by three histidines and the COO- of the carboxyl terminus. The coordination geometry is nonregular and appears to be a distorted octahedron in which two adjacent positions are not occupied by ligands. Two cavities, in the shapes of a bent cylinder and a frustum, connect the unoccupied positions to the surface of the enzyme. The iron, with two adjacent and unoccupied positions, is poised to interact with the 1,4-diene system of the substrate and with molecular oxygen during catalysis.

Nudix hydrolases catalyze the hydrolysis of n ucleoside d iphosphates linked to other moieties, X , and contain the sequence motif or Nudix box, GX 5 EX 7 REUXEEXGU. The mechanisms of Nudix hydrolases are highly diverse in the position on the substrate at which nucleophilic substitution occurs, and in the number of required divalent cations. While most proceed by associative nucleophilic substitutions by water at specific internal phosphorus atoms of a diphosphate or polyphosphate chain, members of the GDP-mannose hydrolase sub-family catalyze dissociative nucleophilic substitutions, by water, at carbon. The site of substitution is likely determined by the positions of the general base and the entering water. The rate accelerations or catalytic powers of Nudix hydrolases range from 10 9 - to 10 12 -fold. The reactions are accelerated 10 3 –10 5 -fold by general base catalysis by a glutamate residue within, or beyond the Nudix box, or by a histidine beyond the Nudix box. Lewis acid catalysis, which contributes ⩾10 3 –10 5 -fold to the rate acceleration, is provided by one, two, or three divalent cations. One divalent cation is coordinated by two or three conserved residues of the Nudix box, the initial glycine and one or two glutamate residues, together with a remote glutamate or glutamine ligand from beyond the Nudix box. Some Nudix enzymes require one (MutT) or two additional divalent cations (Ap 4 AP), to neutralize the charge of the polyphosphate chain, to help orient the attacking hydroxide or oxide nucleophile, and/or to facilitate the departure of the anionic leaving group. Additional catalysis (10–10 3 -fold) is provided by the cationic side chains of lysine and arginine residues and by H-bond donation by tyrosine residues, to orient the general base, or to promote the departure of the leaving group. The overall rate accelerations can be explained by both independent and cooperative effects of these catalytic components.

We report the characterization of 5-methoxy-1,2-dimethyl-3-[(4-nitrophenoxy)methyl]indole-4,7-dione (ES936) as a mechanism-based inhibitor of NQO1. Inactivation of NQO1 by ES936 was time- and concentration-dependent and required the presence of a pyridine nucleotide cofactor consistent with a need for metabolic activation. That ES936 was an efficient inhibitor was demonstrated in these studies by the low partition ratio (1.40 +/- 0.03). The orientation of ES936 in the active site of NQO1 was examined by X-ray crystallography and found to be opposite to that observed for other indolequinones acting as substrates. ES936 was oriented in such a manner that, after enzymatic reduction and loss of a nitrophenol leaving group, a reactive iminium species was located in close proximity to nucleophilic His 162 and Tyr 127 and Tyr 129 residues in the active site. To determine if ES936 was covalently modifying NQO1, ES936-treated protein was analyzed by electrospray ionization liquid chromatography/mass spectrometry (ESI-LC/MS). The control NQO1 protein had a mass of 30864 +/- 6 Da (n = 20, theoretical, 30868.6 Da) which increased by 217 Da after ES936 treatment (31081 +/- 7 Da, n = 20) in the presence of NADH. The shift in mass was consistent with adduction of NQO1 by the reactive iminium derived from ES936 (M + 218 Da). Chymotryptic digestion of the protein followed by LC/MS analysis located a tetrapeptide spanning amino acids 126-129 which was adducted with the reactive iminium species derived from ES936. LC/MS/MS analysis of the peptide fragment confirmed adduction of either Tyr 127 or Tyr 129 residues. This work demonstrates that ES936 is a potent mechanism-based inhibitor of NQO1 and may be a useful tool in defining the role of NQO1 in cellular systems and in vivo.

Introduction Quinones are ubiquitous dietary compounds. Reduction of quinones by one-electron reduction systems (cytochrome P450 reductases) results in the formation of reactive semiquinones that can be re-oxidized to quinones by molecular oxygen with the formation of superoxide radical (02-*). Superoxide can lead to the formation of other reactive oxygen species, including the highly mutagenic and carcinogenic hydroxyl radical. In addition, the presence of quinones and their one-electron reduction products can result in the depletion of reduced thiol species. Cytosolic quinone reductase (QR) 1 provides a pathway that obviates all these deleterious reactions. Reduction of quinones via the obligatory two-electron reduction carried out by QR results in the formation of hydroquinones that can be glucuronidated and readily excreted. QR1 [NAD(P)H : (quinone acceptor) oxidoreductase; EC 1.6.99.21 is a widely distributed, predominantly cytosolic, FAD-containing flavoprotein that promotes the obligatory two-electron reduction of many quinones and quinoneimines. (The enzyme has also been referred to as menadione reductase, DT-diaphorase and vitamin-K reductase). It uses NADH or NADPH as reductants with equal facility and is potently inhibited by dicoumarol and similar anticoagulants. Furthermore, QR activity is elevated in many animal tissues and cell lines by addition of xenobiotics. The discovery, purification, molecular characteristics, induction and other properties of this enzyme have been presented previously [ 1,2]. The purification and properties of another mammalian cytosolic FAD-dependent flavoprotein was described in the early 1960's by Liao et al. [3]. This enzyme was able to catalyse the oxidation of reduced N-ribosyland N-alkylnicotinamides by menadione and other quinones. Recently, the determination of the sequence indicated that this enzyme is highly homologous with QR1 [4,5], leading to the conclusion that it is

Crystals of lipoxygenase-1 from soybeans have been grown by the method of vapor diffusion in the presence of sodium formate, ammonium acetate, and lithium chloride at pH 7.0. This enzyme contains a non-heme iron and is closely related to a human lipoxygenase found in leukocytes that participates in the biosynthesis of leukotrienes and lipoxins. The crystals are monoclinic space group C2 with cell dimensions of a = 183.8 A, b = 123.2 A, c = 94.3 A and beta = 102.9 degrees. They diffract beyond 2.7 A, are stable for several days in the x-ray beam, and appear to be suitable for x-ray diffraction studies.

Publisher Summary The Na+/I- symporter (NIS) is a key plasma membrane protein that catalyzes the active accumulation of iodide (I-) in the thyroid gland, that is, the first and critical rate-limiting step in the biosynthesis of the thyroid hormones. NIS is located in the basolateral membrane of the hormone-producing thyroid follicular cells or thyrocytes. NIS is one of at least four major proteins that appear to be distinctively thyroidal, the other three being Tg, TPO and the TSH receptor. Each of these four proteins mediates a crucial step in thyroid hormogenesis. Each of them also plays an important role in thyroid function. They are also considered thyroid-specific markers. This chapter presents the rat NIS cDNA clone and discusses a secondary structure model for the NIS molecule. The product of the NIS cDNA clone is sufficient to elicit Na+/I- symport activity in both oocytes and mammalian cells, suggesting that NIS probably functions as a single subunit or as an oligomer of identical subunits. The role of NIS as the mediator of I- accumulation is consistent with its placing alongside other Na+-dependent anion transporters based on clustering relationships between its deduced amino acid sequence and the deduced sequences of other transporters. A comparison of the predicted amino acid sequence of NIS with those of other cloned Na+-dependent cotransporters revealed the highest degree of homology.

It has been proposed that during ATP synthesis/hydrolysis F1 ATPases experience a complex pattern of nucleotide binding and release during the catalytic cycle (binding change mechanism). This type of mechanism has implications that can be correlated with the structure of the enzyme. F1-ATPases (stoichiometry alpha 3 beta 3 gamma delta epsilon) are essentially a symmetrical trimer of pairs of the major subunits (alpha and beta); the minor subunits (gamma, delta and epsilon) are in single copies and interact with the trimer in an asymmetrical fashion. The asymmetry introduced by the minor subunits has important structural and functional consequences: (1) it introduces differences between the potentially equivalent binding and catalytic sites in the major subunits, (2) it restricts the ways in which a binding change mechanism can occur, and (3) it governs the way in which the F1 interacts with the (asymmetrical) F0 sector.

Genetic and sequence information are reported for an angiotensin II-reactive antibody (Ab1, MAb 110) and an anti--anti-idiotypic antibody (Ab3, MAb 131) that have identical antigen binding properties and that are related by an anti-idiotypic antibody (Ab2-beta) that satisfies accepted biochemical criteria for an internal image-bearing antibody. The sequences of the variable regions of the Ab3 and of the Ab1 are nearly identical, even though the Ab1 is an antibody to a peptide and the Ab3 is an antibody to a globular protein. Significantly, amino acid residues that make critical contacts with antigen in the crystal structure of the Ab3-antigen complex are highly conserved in Ab1, suggesting that the epitopes of the Ab2-beta recognized by the Ab3 do indeed resemble the bound structure of the antigen.

The three-dimensional structure of the pFc′ fragment of guinea pig IgGl, a dimer of C H 3 homology sub-units, has been determined to 3.125 A nominal resolution by crystallographic analysis. The crystal structure was solved by molecular replacement methods using as a probe the refined structure of a human IgG1 C H 1 homology sub-unit. The path of the polypeptide chain has been traced from Gly341 to Ile441. No continuous electron density was found for the N-terminal segment before Gly341. The majority of the amino acid side chains were located in the electron density map. The overall tertiary structure and many features of secondary structure found in other constant region homology sub-units of immunoglobulins are preserved in pFc′. The high homology in amino acid sequence among C H 3 regions of different immunoglobulins can be partly explained on the basis of the three-dimensional structure; residues which are involved in contacts essential to the tertiary and quaternary structure of the molecule are conserved or conservatively replaced. Amino acid sequence differences in the C H 3 regions of different sub-classes of IgG are analyzed with respect to their position in the three-dimensional structure and the effector function properties of the different sub-classes.

The theory of fusion of molecular crystals with orientational degrees of freedom developed by Pople and Karasz [1] is extended by taking into account the existence of more than two possible positions of minimum orientational energy in the crystal. Now the model has two parameters: D , the number of these possible positions; and as before, v , a measure of the relative energy barriers for the rotation of a molecule and for its diffusion to an interstitial site. The main features derived from the model, when D > 2, are (1) solid-solid and melting transitions are always of first order; (2) the values of v at which solid-solid transition and fusion temperatures ( T t and T m ) coalesce increase when D increases; (3) solid-solid transition entropies increase with D and for each D , with v ; (4) at constant v there is a decrease of T t and of T m when D increases; (5) relative volume changes on melting become smaller than the relative volume changes at the solid-solid transition when v > 0.26, this change becomes as small as 4 per cent for D = 60 and v = 0.5. The quantitative predictions of the model are compared with experimental results for plastic crystals. There is good agreement between theory and experiment. For solid-solid transitions the agreement is much better than with the calculations of Pople and Karasz, as would be expected from the fact that in the present theory D is a parameter.

The esteroproteolytic enzyme, a serine protease from porcine pancreas, has been crystallized and characterized by X-ray diffraction. Two closely related crystal forms were observed. The unit cell dimensions of the first form are a = 59.2 A, b = 96.4 A and c = 47.4 A, space group P21212 with one molecule (mol. wt 30,000) per asymmetric unit. The unit cell dimensions of the second form are a = 59.2 A, b = 96.4 A and c = 94.8 A, space group P212121. Mercury derivatives of enzyme inhibitors were used to obtain multiple isomorphous heavy-atom substituents suitable for phase determination.

One of the central problems in immunochemistry is that of defining the structural basis for the activity, specificity, and physiological function of antibody molecules. Although several experimental approaches such as amino acid sequence determination and affinity labeling have provided important clues to the solution of this problem, it is generally accepted that X-ray crystallographic analysis is the only technique currently available to reveal the complete three-dimensional structure of proteins. In recent years, several laboratories have succeeded in obtaining atomic-resolution models of immunoglobulins (Ig’s) by X-ray diffraction methods. A review of the major conclusions achieved in these studies is the aim of this chapter.

The conformation of the interchain disulfide bond between the light and the heavy chains of human immunoglobulin G4 (IgG4) was modeled based on the known structure of a human IgG1 Fab. Exploration of a large number of conformations followed by energy minimization using molecular mechanics methods rendered a plausible model for the disulfide. In this model the disulfide adopts the long, trans-gauche-trans configuration found in immunoglobulin intrachain bridges and not the conformations most commonly observed in other proteins (left-handed spiral or right-handed hook). Heavy chain residues H217 and H218 pack tightly against the disulfide and put restrictions on which sequences can exist in the proposed conformation. Sequence analysis at these positions shows that: (a) all human IgG4 and IgG3 are compatible with the model; (b) IgG2 and human immunoglobulins of other classes cannot adopt the conformation proposed for IgG4.

The structure of human immunoglobulin Fab′ fragment has been elucidated at a resolution of 6 A. There are two globular subunits, which presumably correspond to the variable and constant regions of the heavy and light polypeptide chains of immunoglobulins

The structure of the Fab' fragment of a human myeloma protein (IgG1 (λ) New) has been determined by X-ray crystallographic analysis to a nominal resolution of 0.2 nm. Each of the structure subunits corresponding to the variable and to the constant homology regions of the light and heavy polypeptide chains contains two irregular (3-sheets which are roughly parallel to each other and surround a tightly packed interior of hydrophobic side chains. The regions of the hypervariable sequences in the light and heavy chains occur in close spatial proximity at one end of the molecule, defining the active site of IgG New. The role of these hypervariable regions in defining the size and shape of the active site of different immunoglobulins is discussed on the basis of the three-dimensional model of Fab' New. Several ligands that bind to the active centre of IgG New have been used to obtain crystalline ligand-Fab' New complexes which were investigated by difference Fourier maps. These studies are analysed in terms of the biological function and specificity of antibodies.