Gregory A. Grant

The ACT Domain: A Small Molecule Binding Domain and Its Role as a Common Regulatory Element

The ACT domain is a structural motif in proteins of 70–80 amino acids that is one of a growing number of different intracellular small molecule binding domains that function in the control of metabolism, solute transport, and signal transduction (1–5). The first structure of an ACT domain was determined in 1995 with the crystal structure of Escherichia coli D-3phosphoglycerate dehydrogenase (6), a tetrameric protein containing one ACT domain per subunit. This structure represents the archetypical ACT domain and is composed of four strands and two helices arranged in a fold as shown in Fig. 1. However, it was not recognized as a recurring motif until 1999whenAravind andKoonin (2) proposed this based on a PSI-Blast (position-specific iterating-Blast) sequence data base search using the small subunit (IlvN) of acetolactate synthase. This search identified a diverse group of proteins that were noted to be involved in someway in amino acid and purine metabolism and were regulated by specific amino acids. They named this proposed domain the ACT domain after the first letters of three of the proteins, aspartate kinase-chorismate mutase-tyrA (prephenate dehydrogenase). In 2001, the structure of the Lrp-like transcriptional regulator fromPyrococcus furiosiswas published (7). This was the first structure of a transcription factor that contained an ACT domain. A PSI-Blast analysis of its sequence by Ettema et al. (3) revealed an additional group of proteins, including both enzymes and transcription regulators, that they proposed contained a novel type of ACT domain, which they named the RAMdomain for regulator of amino acidmetabolism. The Lrplike transcriptional regulator contains the ACT domain fold ( ), but the sequence alignment resulting from the PSIBlast search appeared to reveal a somewhat different pattern of conservation of residue type (3) (Fig. 2). Mutagenesis data also suggested that the ligand binding sites of the Lrp-like protein were located differently than in the ACT domains described previously. Although the ACT domain from E. coli D-3-phosphoglycerate dehydrogenase and the Lrp-like transcription factor superimpose very well (1.8-Å root mean square deviation), the dimer interfaces of each are significantly different. The ACT domain dimers of phosphoglycerate dehydrogenase form a side-by-side structure producing an extended 8-stranded sheet (Fig. 3A). On the other hand, the sheets of the ACT domain dimers of the Lrp transcription factor assume a more face-to-face configuration (Fig. 3J). These observations formed the basis for the proposed division into ACT and RAM domains. As will be seen later in this review, recently determined structures demonstrate that the ACT domain shows an increasing diversity in tertiary and quaternary architecture as well as ligand binding interactions. A novel type of ACT domain-containing protein family whose members contain ACT domain repeats has also been identified by sequence analysis in Arabadopsis (8). These proteins were termed ACR proteins. There are at least 8 genes in the 5 chromosomes of Arabadopsis that belong to the “ACR” protein family. Proteins similar to the ACR family have also been identified in rice (Oryza sativa) (9). The majority of ACT domain-containing proteins appear to interact with amino acids and are involved in some aspect of regulation of amino acid metabolism (2–4) (Fig. 1). These include both metabolic enzymes and transcription regulators. In fact, the expression of some ACT-containing enzymes is under the control of ACT-containing transcription regulators. This has resulted in the ACT domain being referred to as “the regulatory domain in amino acid metabolism” in the SCOP (structural classification of proteins) data base. However, notable exceptions to this generality have been revealed in recent years. These include the NikR transcriptional regulator that binds nickel and functions in the regulation of intracellular nickel levels (10) and the YkoF protein (11) that binds thiamine and is thought to be involved in thiamine transport. It is noteworthy that the 80–90-amino acid long ribonucleoprotein motif of RNA binding proteins (12, 13) also possesses the same fold as the ACT domain. These domains bind RNA through interaction at the face of their -sheet structure rather than binding smallmolecules in loop regions like the ACT domains. It is not known how these very similar domains may be related evolutionarily, but their similarity is intriguing. The RNA binding domains have their own unique pattern of conserved consensus sequences and the PSI-Blast searches that identified the ACT and RAM domains did not appear to select any RNA binding domains.

Increased collagen cross-linkages in experimental diabetes: reversal by beta-aminopropionitrile and D-penicillamine.

The effects of diabetes on collagen cross-link formation and solubility were investigated in granulation tissue collagen induced by polyester fabric implanted subcutaneously in rats at the same time diabetes was produced by injection of streptozotocin. Thus, all the collagen analyzed was formed in a diabetic milieu. Ten days later the implants were removed and the total collagen content as well as the fraction soluble in 0.5 M acetic acid was determined. Predominantly type I collagen accumulated in the implants. Total collagen content was the same in diabetics and controls; however, the acid-soluble fraction in diabetic animals was only half that of controls (8.5% and 17.7%, respectively), and the ratio of β chains to a chains in the acid-soluble fraction was higher in diabetics (0.89) than in controls (0.69). In animals treated with β-aminopropionitrile or D-penicillamine the acid-soluble fraction of collagen from diabetics equaled that from controls. These observations indicate that both intramolecular and intermolecular cross-links are increased in type I collagen from diabetic animals. Since these cross-links interfere with degradation of collagen by collagenase, they may contribute to accelerated intimal sclerosis of arteries and to capillary basement membrane thickening in diabetes.

A new family of 2-hydroxyacid dehydrogenases

The NADH-dependent hydroxypyruvate reductase from cucumber and the pdxB gene product of E. coli display significant homology to E. coli D-3-phosphoglycerate dehydrogenase. In contrast, these proteins do not display much similarity with other oxidoreductases or with other 2-hydroxyacid dehydrogenases in particular. On the basis of their relatedness and the structure of their substrates, these three enzymes constitute a new family of 2-hydroxyacid dehydrogenases distinct from malate and lactate dehydrogenase.

Crystal structure of Mycobacterium tuberculosis D-3-phosphoglycerate dehydrogenase : Extreme asymmetry in a tetramer of identical subunits

Phosphoglycerate dehydrogenases exist in at least three different structural motifs. The first d-3-phosphoglycerate dehydrogenase structure to be determined was from Escherichia coli and is a tetramer composed of identical subunits that contain three discernable structural domains. The crystal structure of d-3-phosphoglycerate dehydrogenase from Mycobacterium tuberculosis has been determined at 2.3 Å. This enzyme represents a second structural motif of the d-3-phosphoglycerate dehydrogenase family, one that contains an extended C-terminal region. This structure is also a tetramer of identical subunits, and the extended motif of 135 amino acids exists as a fourth structural domain. This intervening domain exerts quite a surprising characteristic to the structure by introducing significant asymmetry in the tetramer. The asymmetric unit is composed of two identical subunits that exist in two different conformations characterized by rotation of ∼180° around a hinge connecting two of the four domains. This asymmetric arrangement results in the formation of two different and distinct domain interfaces between identical domains in the asymmetric unit. As a result, the surface of the intervening domain that is exposed to solvent in one subunit is turned inward in the other subunit toward the center of the structure where it makes contact with other structural elements. Significant asymmetry is also seen at the subunit level where different conformations exist at the NAD-binding site and the putative serine-binding site in the two unique subunits.

Isolation of the fibrinogen-binding region of platelet thrombospondin

Purified platelet thrombospondin binds to immobilized fibrinogen if both Ca++ and Mg++ are present. Digestion of the purified molecule with thermolysin results in a limited number of discrete proteolytic fragments. When such digests are subjected to affinity chromatography on immobilized fibrinogen, only the fragments with Mr of 120,000 and 140,000 are specifically bound and subsequently eluted by the addition of EDTA to the column buffer. Examination by SDS-PAGE under both reducing and nonreducing conditions reveals that the fibrinogen-binding domain is derived from the region of the thrombospondin molecule containing the interchain disulfide bonds. The requirement for Ca++ and Mg++ for optimal binding to fibrinogen is also manifest by the Mr 120,000/140,000 thermolytic fragments.

Glycosylation of Human Glomerular Basement Membrane Collagen: Increased Content of Hexose in Ketoamine Linkage and Unaltered Hydroxylysine-O-Glycosides in Patients with Diabetesd

To study the glycosylation of glomerular basement membrane collagen (GBMC) in diabetes, kidneys were obtained at autopsy from 5 patients with insulin-requiring diabetes of long duration and diabetic complications, and from 5 control subjects. Glomeruli were prepared by sieving and collagen was isolated by limited pepsin proteolysis followed by salt precipitations. Amino acid analyses of the collagen preparations, after acid hydrolysis, indicated a composition consistent with that of type IV collagen. No differences in the relative contents of various amino acids, and in particular, 3-hydroxyproline, 4-hydroxyproline and hydroxylysine, were noted between diabetic and control samples. Non-enzymatic glucosylation was assessed by measuring hexose in ketoamine linkage with thiobarbituric acid after conversion to 5-hydroxymethylfurfural. In 4 of the 5 patients studied, glucosylation values exceeded the mean +2 S.D. of the controls; in the fifth subject glucosylation was in the high normal range. No correlation between the severity of diabetes and hexose content of GBMC was noted, however. In further studies, enzymatic glycosylation of GBMC was assayed after alkaline hydrolysis by separation of glucosylgalactosyl-O-hydroxylysine, galactosyl-O-hydroxylysine, and unsubstituted hydroxylysine in an amino acid analyzer. No differences in the relative contents of hydroxylysine-O-glycosides were evident between diabetic and control GBMC. The results suggest that non-enzymatic glucosylation, but not glycosylation catalyzed by collagen glucosyl and galactosyl transferases, is increased in diabetes. The increased carbohydrate content of collagen may lead to decreased turnover and/or excessive accumulations of basement membrane collagen thus contributing to the vascular complications of diabetes.

Binding of native κ-neurotoxins and site-directed mutants to nicotinic acetylcholine receptors

The κ-neurotoxins are useful ligands for the pharmacological characterization of nicotinic acetylcholine receptors because they are potent antagonists at only a subgroup of these receptors containing either α3- or α4-subunits (IC50 ≤ 100 nM). Four of these highly homologous, 66 amino acid peptides have been purified from the venom of Bungarus multicinctus [κ-bungarotoxin (κ-Bgt), κ2-Bgt, κ3-Bgt] and Bungarus flaviceps [κ-flavitoxin (κ-Fvt)]. Two approaches were taken to examine the binding of these toxins to nicotinic receptors. First, venom-derived κ-Fvt and κ-Bgt were radioiodinated and the specific binding was measured of these toxins to overlapping synthetic peptides (16–20 amino acids in length) prepared based on the known sequence of the nicotinic receptor α3-subunit. At least two main regions of interaction between the toxins and the receptor subunit were identified, both lying in the N-terminal region of the subunit that is exposed to the extracellular space. The second approach examined the importance of several sequence positions in κ-Bgt for binding to α3-containing receptors in autonomic ganglia and α1-containing muscle receptors. This was done using site-directed mutants of κ-Bgt produced by an Escherichia coli expression system. Arg-34 and position 36 were important for binding to both receptor subtypes, while replacing Gln-26 with Trp-26 (an invariant in α-neurotoxins) increased affinity for the muscle receptor by 8-fold. The results confirm that κ-neurotoxins bind potently to the α3-subunit and bind with considerably reduced affinity (Kd ≈ 10 μM) to muscle receptors. Site-directed mutagenesis of recombinant κ-Bgt is thus an important approach for the study of structure-function relationships between κ-Bgt and nicotinic receptors.

D-3-Phosphoglycerate Dehydrogenase from Mycobacterium tuberculosis Is a Link between the Escherichia coli and Mammalian Enzymes

d-3-Phosphoglycerate dehydrogenase (PGDH) from Mycobacterium tuberculosis has been isolated to homogeneity and displays an unusual relationship to the Escherichia coli and mammalian enzymes. In almost all aspects investigated, the M. tuberculosis enzyme shares the characteristics of the mammalian PGDHs. These include an extended C-terminal motif, substrate inhibition kinetics, dependence of activity levels and stability on ionic strength, and the inability to utilize α-ketoglutarate as a substrate. The unique property that the M. tuberculosis enzyme shares with E. coli PGDH that it is very sensitive to inhibition by l-serine, with an I0.5 = 30 μm. The mammalian enzymes are not inhibited by l-serine. In addition, the cooperativity of serine inhibition appears to be modulated by chloride ion, becoming positively cooperative in its presence. This is modulated by the gain of cooperativity in serine binding for the first two effector sites. The basis for the chloride modulation of cooperativity is not known, but the sensitivity to serine inhibition can be explained in terms of certain amino acid residues in critical areas of the structures. The differential sensitivity to serine inhibition by M. tuberculosis and human PGDH may open up interesting possibilities in the treatment of multidrug-resistant tuberculosis.

Modification of Cysteine

This unit describes a number of methods for modifying cysteine residues of proteins and peptides by reduction and alkylation procedures. A general procedure for alkylation of cysteine residues in a protein of known size and composition with haloacyl reagents or N‐ethylmaleimide (NEM) is presented, and alternate protocols describe similar procedures for use when the size and composition are not known and when only very small amounts of protein are available. Alkylations that introduce amino groups using bromopropylamine and N‐(iodoethyl)‐trifluoroacetamide are also presented. Two procedures that are often used for subsequent sequence analysis of the protein, alkylation with 4‐vinylpyridine and acrylamide, are described, and a specialized procedure for 4‐vinylpyridine alkylation of protein that has been adsorbed onto a sequencing membrane is also presented. Reversible modification of cysteine residues by way of sulfitolysis is described, and a protocol for oxidation with performic acid for amino acid compositional analysis is also provided. Gentle oxidation of cysteine residues to disulfides by exposure to air is detailed. Support protocols are included for recrystallization of iodoacetic acid, colorimetric detection of free sulfhydryls, and desalting of modified samples.

[53] Collagenolytic protease from fiddler crab (Uca pugilator)

Publisher Summary This chapter presents the procedure for purification and assaying of crab collagenolytic protease. The procedure described in the chapter uses the glands from 8000 crabs, received in shipments of 1000 each. The purification steps are following: preparation of acetone powder; first gel filtration; ion-exchange chromatography; hydroxyapatite chromatography; second gel filtration. This procedure produces approximately 100-150 mg of homogeneous crab protease from the glands of 8000 fiddler crabs. The enzyme is stable for several months in slightly acidic solution (pH -6.0) at –20°C, and can be stored indefinitely as a lyophilized powder. Crab collagenase can be assayed specifically for collagenolytic activity with a standard assay, which quantitates the release of soluble [ 14 C]glycine-containing peptides from native reconstituted guinea pig skin collagen fibrils, or spectrophotometrically by following esterase or amidase activity with synthetic substrates.