Mutagenesis of the regulatory domain of phenylalanine hydroxylase

The regulatory domain of phenylalanine hydroxylase (PAH, EC 1.14.16.1) consists of more than 100 amino acids at the N terminus, the removal of which significantly activates the enzyme. To study the regulatory properties controlled by the N terminus, a series of truncations and site-specific mutations were made in this region of rat PAH. These enzymes were expressed highly in Escherichia coli and purified through a pterin-conjugated Sepharose affinity column. The removal of the first 26 amino acids of the N terminus increased the activity by about 20-fold, but removal of the first 15 amino acids increased the activity by only 2-fold. Replacing serine-29 of rat PAH with cysteine from the same site of human PAH increased the activity by more than 4-fold. Mutation of serine to other amino acids with varying side chains: alanine, methionine, leucine, aspartic acid, asparagine, and arginine also resulted in significant activation, indicating a serine-specific inhibitory effect. But these site-specific mutants showed 30–40% lower activity when assayed with 6-methyl-5,6,7,8-tetrahydropterin. Stimulation of hydroxylase activity by preincubation of the enzyme with phenylalanine was inversely proportional to the activation state of all these mutants. Combined with recent crystal structures of PAH [Kobe, B. et al. (1999) Nat. Struct. Biol. 6, 442–448; and Erlandsen, H., Bjorgo, E., Flatmark, T. & Stevens, R. C. (2000) Biochemistry 39, 2208–2217], these data suggest that residues 16–26 have a controlling regulatory effect on the activity by interaction with the dihydroxypropyl side chain of (6R)-5,6,7,8-tetrahydrobiopterin. The serine/cysteine switch explains the difference in regulatory properties between human and rat PAH. The N terminus as a whole is important for maintaining rat PAH in an optimum catalytic conformation.

Polar residues drive association of polyleucine transmembrane helices

Although many polar residues are directly involved in transmembrane protein functions, the extent to which they contribute to more general structural features is still unclear. Previous studies have demonstrated that asparagine residues can drive transmembrane helix association through interhelical hydrogen bonding [Choma, C., Gratkowski, H., Lear, J. D. & DeGrado, W. F. (2000) Nat. Struct. Biol. 7, 161–166; and Zhou, F. X., Cocco, M. J., Russ, W. P., Brunger, A. T. & Engelman, D. M. (2000) Nat. Struct. Biol. 7, 154–160]. We have studied the ability of other polar residues to promote helix association in detergent micelles and in biological membranes. Our results show that polyleucine sequences with Asn, Asp, Gln, Glu, and His, residues capable of being simultaneously hydrogen bond donors and acceptors, form homo- or heterooligomers. In contrast, polyleucine sequences with Ser, Thr, and Tyr do not associate more than the polyleucine sequence alone. The results therefore provide experimental evidence that interactions between polar residues in the helices of transmembrane proteins may serve to provide structural stability and oligomerization specificity. Furthermore, such interactions can allow structural flexibility required for the function of some membrane proteins.

Structure of a flavin-binding plant photoreceptor domain: Insights into light-mediated signal transduction

Phototropin, a major blue-light receptor for phototropism in seed plants, exhibits blue-light-dependent autophosphorylation and contains two light, oxygen, or voltage (LOV) domains and a serine/threonine kinase domain. The LOV domains share homology with the PER-ARNT-SIM (PAS) superfamily, a diverse group of sensor proteins. Each LOV domain noncovalently binds a single FMN molecule and exhibits reversible photochemistry in vitro when expressed separately or in tandem. We have determined the crystal structure of the LOV2 domain from the phototropin segment of the chimeric fern photoreceptor phy3 to 2.7-Å resolution. The structure constitutes an FMN-binding fold that reveals how the flavin cofactor is embedded in the protein. The single LOV2 cysteine residue is located 4.2 Å from flavin atom C(4a), consistent with a model in which absorption of blue light induces formation of a covalent cysteinyl-C(4a) adduct. Residues that interact with FMN in the phototropin segment of the chimeric fern photoreceptor (phy3) LOV2 are conserved in LOV domains from phototropin of other plant species and from three proteins involved in the regulation of circadian rhythms in Arabidopsis and Neurospora. This conservation suggests that these domains exhibit the same overall fold and share a common mechanism for flavin binding and light-induced signaling.

Identification of two residues in MCM5 critical for the assembly of MCM complexes and Stat1-mediated transcription activation in response to IFN-γ

In response to IFN-γ, the latent cytoplasmic Stat1 (signal transducer and activator of transcription) proteins translocate into the nucleus and activate transcription. We showed previously that Stat1 recruits a group of nuclear proteins, among them MCM5 (minichromosome maintenance) and MCM3, for transcription activation. MCM5 directly interacts with the transcription activation domain (TAD) of Stat1 and enhances Stat1-mediated transcription activation. In this report, we identified two specific residues (R732, K734) in MCM5 that are required for the direct interaction between Stat1 and MCM5 both in vitro and in vivo. MCM5 containing mutations of R732/K734 did not enhance Stat1-mediated transcription activation in response to IFN-γ. In addition, it also failed to form complexes with other MCM proteins in vivo, suggesting that these two residues may be important for an interaction domain in MCM5. Furthermore, MCM5 bearing mutations in its ATPase and helicase domains did not enhance Stat1 activity. In vitro binding assays indicate that MCM3 does not interact directly with Stat1, suggesting that the presence of MCM3 in the group of Stat1TAD-interacting proteins is due to the association of MCM3 with MCM5. Finally, gel filtration analyses of nuclear extracts from INF-γ-treated cells demonstrate that there is a MCM5/3 subcomplex coeluting with Stat1. Together, these results strongly suggest that Stat1 recruits a MCM5/3 subcomplex through direct interaction with MCM5 in the process of IFN-γ-induced gene activation.

Peptide hybrids containing α- and β-amino acids: Structure of a decapeptide β-hairpin with two facing β-phenylalanine residues

A β-hairpin conformation has been characterized in crystals of the decapeptide t-butoxycarbonyl-Leu-Val-βPhe-Val-DPro-Gly-Leu-βPhe-Val-Val-methyl ester [βPhe; (S)-β3 homophenylalanine] by x-ray diffraction. The polypeptide chain reversal is nucleated by the centrally positioned DPro-Gly segment, which adopts a type-I′ β-turn conformation. Four intramolecular cross-strand hydrogen bonds stabilize the peptide fold. The βPhe(3) and βPhe(8) residues occupy facing positions on the hairpin, with the side chains projecting on opposite faces of the β-sheet. At the site of insertion of β-residues, the polarity of the peptide units along each strand reverses, as compared with the α-peptide segments. In this analog, a small segment of a polar sheet is observed, where adjacent CO and NH groups line up in opposite directions in each strand. In the crystal, an extended β-sheet is formed by hydrogen bonding between strands of antiparallel pairs of β-hairpins. The crystallographic parameters for C65H102N10O13⋅ 3H2O are: space group P212121; a = 19.059(8) Å, b = 19.470(2) Å, c = 21.077(2) Å; Z = 4; agreement factor R1 = 9.12% for 3,984 data observed >4σ(F) and a resolution of 0.90 Å.

The interacting domains of three MutL heterodimers in man: hMLH1 interacts with 36 homologous amino acid residues within hMLH3, hPMS1 and hPMS2

In human cells, hMLH1, hMLH3, hPMS1 and hPMS2 are four recognised and distinctive homologues of MutL, an essential component of the bacterial DNA mismatch repair (MMR) system. The hMLH1 protein forms three different heterodimers with one of the other MutL homologues. As a first step towards functional analysis of these molecules, we determined the interacting domains of each heterodimer and tried to understand their common features. Using a yeast two-hybrid assay, we show that these MutL homologues can form heterodimers by interacting with the same amino acid residues of hMLH1, residues 492–742. In contrast, three hMLH1 partners, hMLH3, hPMS1 and hPMS2 contain the 36 homologous amino acid residues that interact strongly with hMLH1. Contrary to the previous studies, these homologous residues reside at the N-terminal regions of three subdomains conserved in MutL homologues in many species. Interestingly, these residues in hPMS2 and hMLH3 may form coiled-coil structures as predicted by the MULTICOIL program. Furthermore, we show that there is competition for the interacting domain in hMLH1 among the three other MutL homologues. Therefore, the quantitative balance of these three MutL heterodimers may be important in their functions.

Treble clef finger—a functionally diverse zinc-binding structural motif

Detection of similarity is particularly difficult for small proteins and thus connections between many of them remain unnoticed. Structure and sequence analysis of several metal-binding proteins reveals unexpected similarities in structural domains classified as different protein folds in SCOP and suggests unification of seven folds that belong to two protein classes. The common motif, termed treble clef finger in this study, forms the protein structural core and is 25–45 residues long. The treble clef motif is assembled around the central zinc ion and consists of a zinc knuckle, loop, β-hairpin and an α-helix. The knuckle and the first turn of the helix each incorporate two zinc ligands. Treble clef domains constitute the core of many structures such as ribosomal proteins L24E and S14, RING fingers, protein kinase cysteine-rich domains, nuclear receptor-like fingers, LIM domains, phosphatidylinositol-3-phosphate-binding domains and His-Me finger endonucleases. The treble clef finger is a uniquely versatile motif adaptable for various functions. This small domain with a 25 residue structural core can accommodate eight different metal-binding sites and can have many types of functions from binding of nucleic acids, proteins and small molecules, to catalysis of phosphodiester bond hydrolysis. Treble clef motifs are frequently incorporated in larger structures or occur in doublets. Present analysis suggests that the treble clef motif defines a distinct structural fold found in proteins with diverse functional properties and forms one of the major zinc finger groups.

Protective Function of Chloroplast 2-Cysteine Peroxiredoxin in Photosynthesis. Evidence from Transgenic Arabidopsis1

2-Cysteine peroxiredoxins (2-CPs) constitute a ubiquitous group of peroxidases that reduce cell-toxic alkyl hydroperoxides to their corresponding alcohols. Recently, we cloned 2-CP cDNAs from plants and characterized them as chloroplast proteins. To elucidate the physiological function of the 2-CP in plant metabolism, we generated antisense mutants in Arabidopsis. In the mutant lines a 2-CP deficiency developed during early leaf and plant development and eventually the protein accumulated to wild-type levels. In young mutants with reduced amounts of 2-CP, photosynthesis was impaired and the levels of D1 protein, the light-harvesting protein complex associated with photosystem II, chloroplast ATP synthase, and ribulose-1,5-bisphosphate carboxylase/oxygenase were decreased. Photoinhibition was particularly pronounced after the application of the protein synthesis inhibitor, lincomycin. We concluded that the photosynthetic machinery needs high levels of 2-CP during leaf development to protect it from oxidative damage and that the damage is reduced by the accumulation of 2-CP protein, by the de novo synthesis and replacement of damaged proteins, and by the induction of other antioxidant defenses in 2-CP mutants.

Oxidative Turnover of Soybean Root Glutamine Synthetase. In Vitro and in Vivo Studies1

Glutamine synthetase (GS) is the key enzyme in ammonia assimilation and catalyzes the ATP-dependent condensation of NH3 with glutamate to produce glutamine. GS in plants is an octameric enzyme. Recent work from our laboratory suggests that GS activity in plants may be regulated at the level of protein turnover (S.J. Temple, T.J. Knight, P.J. Unkefer, C. Sengupta-Gopalan [1993] Mol Gen Genet 236: 315–325; S.J. Temple, S. Kunjibettu, D. Roche, C. Sengupta-Gopalan [1996] Plant Physiol 112: 1723–1733; S.J. Temple, C. Sengupta-Gopalan [1997] In C.H. Foyer, W.P. Quick, eds, A Molecular Approach to Primary Metabolism in Higher Plants. Taylor & Francis, London, pp 155–177). Oxidative modification of GS has been implicated as the first step in the turnover of GS in bacteria. By incubating soybean (Glycine max) root extract enriched in GS in a metal-catalyzed oxidation system to produce the ·OH radical, we have shown that GS is oxidized and that oxidized GS is inactive and more susceptible to degradation than nonoxidized GS. Histidine and cysteine protect GS from metal-catalyzed inactivation, indicating that oxidation modifies the GS active site and that cysteine and histidine residues are the site of modification. Similarly, ATP and particularly ATP/glutamate give the enzyme the greatest protection against oxidative inactivation. The roots of plants fed ammonium nitrate showed a 3-fold increase in the level of GS polypeptides and activity compared with plants not fed ammonium nitrate but without a corresponding increase in the GS transcript level. This would suggest either translational or posttranslational control of GS levels.

Identification of Tyrosine Residues in Constitutively Activated Fibroblast Growth Factor Receptor 3 Involved in Mitogenesis, Stat Activation, and Phosphatidylinositol 3-Kinase Activation

Fibroblast growth factor receptor 3 (FGFR3) mutations are frequently involved in human developmental disorders and cancer. Activation of FGFR3, through mutation or ligand stimulation, results in autophosphorylation of multiple tyrosine residues within the intracellular domain. To assess the importance of the six conserved tyrosine residues within the intracellular domain of FGFR3 for signaling, derivatives were constructed containing an N-terminal myristylation signal for plasma membrane localization and a point mutation (K650E) that confers constitutive kinase activation. A derivative containing all conserved tyrosine residues stimulates cellular transformation and activation of several FGFR3 signaling pathways. Substitution of all nonactivation loop tyrosine residues with phenylalanine rendered this FGFR3 construct inactive, despite the presence of the activating K650E mutation. Addition of a single tyrosine residue, Y724, restored its ability to stimulate cellular transformation, phosphatidylinositol 3-kinase activation, and phosphorylation of Shp2, MAPK, Stat1, and Stat3. These results demonstrate a critical role for Y724 in the activation of multiple signaling pathways by constitutively activated mutants of FGFR3.

Three-dimensional domain swapping in p13suc1 occurs in the unfolded state and is controlled by conserved proline residues

p13suc1 has two native states, a monomer and a domain-swapped dimer. We show that their folding pathways are connected by the denatured state, which introduces a kinetic barrier between monomer and dimer under native conditions. The barrier is lowered under conditions that speed up unfolding, thereby allowing, to our knowledge for the first time, a quantitative dissection of the energetics of domain swapping. The monomer–dimer equilibrium is controlled by two conserved prolines in the hinge loop that connects the exchanging domains. These two residues exploit backbone strain to specifically direct dimer formation while preventing higher-order oligomerization. Thus, the loop acts as a loaded molecular spring that releases tension in the monomer by adopting its alternative conformation in the dimer. There is an excellent correlation between domain swapping and aggregation, suggesting they share a common mechanism. These insights have allowed us to redesign the domain-swapping propensity of suc1 from a fully monomeric to a fully dimeric protein.

Excitation–contraction coupling is unaffected by drastic alteration of the sequence surrounding residues L720–L764 of the α1S II-III loop

The II-III loop of the skeletal muscle dihydropyridine receptor (DHPR) α1S subunit is responsible for bidirectional-signaling interactions with the ryanodine receptor (RyR1): transmitting an orthograde, excitation–contraction (EC) coupling signal to RyR1 and receiving a retrograde, current-enhancing signal from RyR1. Previously, several reports argued for the importance of two distinct regions of the skeletal II-III loop (residues R681–L690 and residues L720–Q765, respectively), claiming for each a key function in DHPR–RyR1 communication. To address whether residues 720–765 of the II-III loop are sufficient to enable skeletal-type (Ca2+ entry-independent) EC coupling and retrograde interaction with RyR1, we constructed a green fluorescent protein (GFP)-tagged chimera (GFP-SkLM) having rabbit skeletal (Sk) DHPR sequence except for a II-III loop (L) from the DHPR of the house fly, Musca domestica (M). The Musca II-III loop (75% dissimilarity to α1S) has no similarity to α1S in the regions R681–L690 and L720–Q765. GFP-SkLM expressed in dysgenic myotubes (which lack endogenous α1S subunits) was unable to restore EC coupling and displayed strongly reduced Ca2+ current densities despite normal surface expression levels and correct triad targeting (colocalization with RyR1). Introducing rabbit α1S residues L720–L764 into the Musca II-III loop of GFP-SkLM (substitution for Musca DHPR residues E724–T755) completely restored bidirectional coupling, indicating its dependence on α1S loop residues 720–764 but its independence from other regions of the loop. Thus, 45 α1S-residues embedded in a very dissimilar background are sufficient to restore bidirectional coupling, indicating that these residues may be a site of a protein–protein interaction required for bidirectional coupling.

Structural aspects of activation pathways of aspartic protease zymogens and viral 3C protease precursors

The three-dimensional structures of the inactive protein precursors (zymogens) of the serine, cysteine, aspartic, and metalloprotease classes of proteolytic enzymes are known. Comparisons of these structures with those of the mature, active proteases reveal that, in general, the preformed, active conformations of the residues involved in catalysis are rendered sterically inaccessible to substrates by the residues of the zymogens’ N-terminal extensions or prosegments. The prosegments interact in nonsubstrate-like fashions with the residues of the active sites in most of the cases. The gastric aspartic proteases have a well-characterized zymogen conversion pathway. Structures of human progastricsin, the inactive intermediate 2, and active human pepsin are known and have been used to define the conversion pathway. The structure of the zymogen precursor of plasmepsin II, the malarial aspartic protease, shows a new twist on the mode of inactivation used by the gastric zymogens. The prosegment of proplasmepsin disrupts the active conformation of the two catalytic aspartic acid residues by inducing a major reorientation of the two domains of the mature protease. The picornaviral 2A and 3C proteases have a chymotrypsin-like tertiary structure but with a cysteine nucleophile. These enzymes cleave themselves from the viral polyprotein in cis (intramolecular cleavage) and carry out trans cleavages of other scissile peptides important for the virus life cycle. Although the structure of the precursor viral polyprotein is unknown, it probably resembles the organization of the proenzymes of the bacterial serine proteases, subtilisin, and α-lytic protease. Cleavage of the prosegment is known to occur in cis for these precursor molecules.

Cysteine protease inhibitors as chemotherapy: Lessons from a parasite target

Papain family cysteine proteases are key factors in the pathogenesis of cancer invasion, arthritis, osteoporosis, and microbial infections. Targeting this enzyme family is therefore one strategy in the development of new chemotherapy for a number of diseases. Little is known, however, about the efficacy, selectivity, and safety of cysteine protease inhibitors in cell culture or in vivo. We now report that specific cysteine protease inhibitors kill Leishmania parasites in vitro, at concentrations that do not overtly affect mammalian host cells. Inhibition of Leishmania cysteine protease activity was accompanied by defects in the parasite’s lysosome/endosome compartment resembling those seen in lysosomal storage diseases. Colocalization of anti-protease antibodies with biotinylated surface proteins and accumulation of undigested debris and protease in the flagellar pocket of treated parasites were consistent with a pathway of protease trafficking from flagellar pocket to the lysosome/endosome compartment. The inhibitors were sufficiently absorbed and stable in vivo to ameliorate the pathology associated with a mouse model of Leishmania infection.

Characterization of a Low-Molecular-Weight Glutenin Subunit Gene from Bread Wheat and the Corresponding Protein That Represents a Major Subunit of the Glutenin Polymer1

Both high- and low-molecular-weight glutenin subunits (LMW-GS) play the major role in determining the viscoelastic properties of wheat (Triticum aestivum L.) flour. To date there has been no clear correspondence between the amino acid sequences of LMW-GS derived from DNA sequencing and those of actual LMW-GS present in the endosperm. We have characterized a particular LMW-GS from hexaploid bread wheat, a major component of the glutenin polymer, which we call the 42K LMW-GS, and have isolated and sequenced the putative corresponding gene. Extensive amino acid sequences obtained directly for this 42K LMW-GS indicate correspondence between this protein and the putative corresponding gene. This subunit did not show a cysteine (Cys) at position 5, in contrast to what has frequently been reported for nucleotide-based sequences of LMW-GS. This Cys has been replaced by one occurring in the repeated-sequence domain, leaving the total number of Cys residues in the molecule the same as in various other LMW-GS. On the basis of the deduced amino acid sequence and literature-based assignment of disulfide linkages, a computer-generated molecular model of the 42K subunit was constructed.