Non-chelating inhibition of the H101N variant of human liver arginase by EDTA

Recombinant wild-type human liver arginase (EC 3.5.3.1) expressed in Escherichia coli was markedly resistant to inhibition by ethylene diamine tetraacetic acid (EDTA). In contrast, half and fully activated species of the H101N variant were totally inactive in the presence of approximately 1 mM EDTA. Dilution of inhibited species in metal-free buffer lead to a time dependent recovery of activity, even when measured in the absence of added Mn2+. The inhibition was mixed type, with predominance of a competitive component (Kii=0.31 mM; Kis=0.022 mM). The structurally related N,N,N',N'-tetramethylethylenediamine was not inhibitory, indicating the importance of the carboxyl groups in EDTA inhibition. We conclude that EDTA inhibition of H101N arginase is not due to interaction with a weakly bound Mn2+ or chelation of essential metal ions.     

Inhibition of human arginase I by substrate and product analogues

Human arginase I is a binuclear manganese metalloenzyme that catalyzes the hydrolysis of l-arginine to generate l-ornithine and urea. We demonstrate that N-hydroxy-l-arginine (NOHA) binds to this enzyme with K_d = 3.6 μM, and nor-N-hydroxy-l-arginine (nor-NOHA) binds with K_d = 517 nM (surface plasmon resonance) or K_d ≈ 50 nM (isothermal titration calorimetry). Crystals of human arginase I complexed with NOHA and nor-NOHA afford 2.04 and 1.55 Å resolution structures, respectively, which are significantly improved in comparison with previously-determined structures of the corresponding complexes with rat arginase I. Higher resolution structures clarify the binding interactions of the inhibitors. Finally, the crystal structure of the complex with l-lysine (K_d = 13 μM) is reported at 1.90 Å resolution. This structure confirms the importance of hydrogen bond interactions with inhibitor α-carboxylate and α-amino groups as key specificity determinants of amino acid recognition in the arginase active site.     

Mutational analysis of Norrin-Frizzled4 recognition

Norrin and Frizzled4 (Fz4) function as a ligand-receptor pair to control vascular development in the retina and inner ear. In mice and humans, mutations in either of the corresponding genes lead to defects in vascular development. The present work is aimed at defining the sequence determinants of binding specificity between Norrin and the Fz4 amino-terminal ligand-binding domain (the "cysteine-rich domain" (CRD)). The principal conclusions are as follows: 1) Norrin binds to the Fz4 CRD and does not detectably bind to the 14 other mammalian Frizzled and secreted Frizzled-related protein CRDs; 2) Norrin and Xenopus Wnt8 recognize largely overlapping regions of the Fz4 CRD; 3) surface determinants on the Fz4 and Fz8 CRDs that allow Norrin to distinguish between these two CRDs reside within several small regions on one face of the CRD; 4) Norrin function depends critically on three pairs of cysteines that form the highly conserved trio of disulfide bonds shared among all cystine knot proteins, but the remaining two putative disulfide bonds are less important; 5) Norrin-CRD binding depends on a largely contiguous group of amino acids in the extended beta-sheet domain of Norrin that are predicted to face away from the interface between the two monomers in the Norrin homodimer; 6) Norrin-CRD binding is strongly modulated by interactions involving charged amino acid side chains; and 7) Norrin-CRD binding is enhanced approximately 10-fold by the addition of heparin. These observations are discussed in the context of Frizzled signaling and the structure and function of other cystine knot proteins.     

Mutational analysis of the human immunodeficiency virus type 1 Vif protein

Lentivirus Vif proteins are potent regulators of virus infectivity. However, relatively little is known about the functional domains, peptide motifs, or residues of any Vif protein. In this report, we present the first extensive mutagenesis analysis of the 192-amino-acid human immunodeficiency virus type 1 (HIV-1) Vif protein. A large number of scanning missense (mostly alanine substitution) and deletion mutations were introduced into the HIV-1HXB3 vif gene, and the resulting proteins were evaluated for the induction of virus infectivity as well as subcellular localization. The results show that amino acids dispersed throughout Vif's linear sequence are important for function. However, because many of the inactive proteins also appear to be mislocalized, we suggest that many of them may actually be misfolded rather lacking an intracellular targeting signal. Interestingly, disruptions within an internal region spanning residues 114 to 146 give rise to mutant proteins that either retain function or are inactive but are not substantially mislocalized. We therefore speculate that this region, which harbors two essential cysteine residues and one essential serine residue, may contain aspects of a putative Vif effector domain.     

Mutational analysis of the engrailed homeodomain recognition helix by phage display.

The homeodomain (HD) is a ubiquitous protein fold that confers DNA binding function on a superfamily of eukaryotic gene regulatory proteins. Here, the DNA binding of recognition helix variants of the HD from the engrailed gene of Drosophila melanogaster was investigated by phage display. Nineteen different combinations of pairwise mutations at positions 50 and 54 were screened against a panel of four DNA sequences consisting of the engrailed consensus, a non-specific DNA control based on the lambda repressor operator OR1 and two model sequence targets con-taining imperfect versions of the 5'-TAAT-3' consensus. The resulting mutant proteins could be divided into four groups that varied with respect to their affinity for DNA and specificity for the engrailed consensus. The altered specificity phenotypes of several mutant proteins were confirmed by DNA mobility shift analysis. Lys50/Ala54 was the only mutant protein that exhibited preferential binding to a sequence other than the engrailed consensus. Arginine was also demonstrated to be a functional replacement for Ala54. The functional combinations at 50 and 54 identified by these experiments recapitulate the distribution of naturally occurring HD sequences and illustrate how the engrailed HD can be used as a framework to explore covariation among DNA binding residues.     

Mutational analysis of human papillomavirus type 16 E7 functions.

The human papillomavirus type 16 E7 gene encodes a nuclear oncoprotein (98 amino acids [AAs] long) consisting of three regions: regions 1 (AAs 1 to 20) and 2 (AAs 21 to 40), which show high homology to the sequences of conserved domains 1 and 2, respectively, of adenovirus E1A; and region 3 (AAs 41 to 98) containing two metal-binding motifs Cys-X-X-Cys (AAs 58 and 91 to 94). We constructed AA deletion (substitution) mutants and single-AA substitution mutants of E7 placed under the control of the simian virus 40 promoter and examined their biological functions. Stable expression of E7 protein in monkey COS-1 cells required almost the entire length of E7 and was markedly lowered by the mutations in region 3. Transactivation of the adenovirus E2 promoter in monkey CV-1 cells was lowered by the mutations. It was abolished by changing Cys-24 to Gly and markedly decreased by a mutation at His-2 or at the metal-binding motifs in region 3. Focal transformation of rat 3Y1 cells by E7 was eliminated by changing His-2 to Asp or Cys-24 to Gly and was greatly impaired by changing Cys-61 or Cys-94 to Gly. The transforming function survived mutations at Leu-13 and Cys-68 and deletion of Asp-Ser-Ser (AAs 30 to 32). The data suggest that regions 1 to 3 are required for its functions and that the meta-binding motifs in region 3 are required to maintain a stable or functional structure of the E7 protein.     

Mutational analysis of human T-cell leukemia virus type 2 Tax.

A mutational analysis of human T-cell leukemia virus type 2 (HTLV-2) Tax (Tax-2) was performed to identify regions within Tax-2 important for activation of promoters through the CREB/ATF or NF-kappaB/Rel signaling pathway. Tax-2 mutations within the putative zinc-binding region as well as mutations at the carboxy terminus disrupted CREB/ATF transactivation. A single mutation within the central proline-rich region of Tax-2 disrupted the transactivation of the NF-kappaB/Rel pathway. Surprisingly, this mutation, which is thought to be in a separate activation domain, was suppressed by mutations within or around the putative zinc-binding region, suggesting an interaction between these two regions. These analyses indicate that the functional regions or domains important for transactivation through the CREB/ATF or NF-kappaB/Rel signaling pathway are similar, but not identical, in Tax-1 and Tax-2. Identification of these distinct Tax-2 mutants should facilitate comparative biological studies of HTLV-1 and HTLV-2 and ultimately lead to the determination of the functional importance of Tax trans-acting capacities in T-lymphocyte transformation by HTLV.     

A kinetic analysis of substrate recognition by uracil-DNA glycosylase from herpes simplex virus type 1

Uracil-DNA glycosylase (UDG) is responsible for the removal of uracil from DNA. It has previously been demonstrated that UDG exhibits some sequence dependence in its activity, although this has not been well characterised. This study has investigated the sequence-dependent activity of UDG from herpes simplex virus type-1 (HSV-1). A more detailed analysis has been possible by using both kinetic and binding assays with a variety of different oligonucleotide substrates. The target uracil has been placed in substrates with either A-T-rich or G-C-rich flanking sequences and analyses have been performed on both the single- and double-stranded forms of each substrate. In the latter the uracil has been placed in both a U·A base pair and a U·G mismatch. It is observed that the sequences flanking the target uracil have a greater effect on UDG activity than the partner base of the uracil. Furthermore, the sequence context effects extend to single-stranded DNA. Systematic examination of the kinetics and binding of UDG with these different substrates has enabled us to examine the origin of the sequence preferences. We conclude that the damage recognition step in the HSV-1 UDG reaction pathway is modulated by local DNA sequence.     

Mutational analysis of human papillomavirus type 11 E5a oncoprotein.

In this study, we investigated the structural basis of human papillomavirus type 11 (HPV-11) E5a transforming activity at the amino acid level. The effects of insertion, deletion , and substitution mutations on teh E5a transforming activity were determined by the assay of anchorage-independent growth. In the conserved Cys-X-Cys structure, substitution of Ser for Cys-73 resulted in indistinguishable transforming activity, whereas substitution of Ser for Cys-75 or Ser for both Cys-73 and Cys-75 retained 50 and 42% transformation, respectively. This suggests that Cys at position 75 may be important for transformation. Charge and structural changes at teh COOH termini of several mutants impaired transformation significantly, but those at the middle region did so only mildly. In addition, the 16,000-molecular-weight pore-forming protein (16K protein) is known to associate with BPV-1, HPV-6, and HPV-16 E5 proteins. In this study, we investigated the correlation between E5a-16K binding affinity and the transforming activity of E5a by the use of 11 E5a mutants. Results show that E5a and these 11 E5a mutants could bind to the 16K protein when these proteins were coexpressed in COS cells, suggesting that simple binding of the 16K protein by E5a may not be sufficient for cell transformation.     

Separating substrate recognition from base hydrolysis in human thymine DNA glycosylase by mutational analysis

Human thymine DNA glycosylase (TDG) was discovered as an enzyme that can initiate base excision repair at sites of 5-methylcytosine- or cytosine deamination in DNA by its ability to release thymine or uracil from G.T and G.U mismatches. Crystal structure analysis of an Escherichia coli homologue identified conserved amino acid residues that are critical for its substrate recognition/interaction and base hydrolysis functions. Guided by this revelation, we performed a mutational study of structure function relationships with the human TDG. Substitution of the postulated catalytic site asparagine with alanine (N140A) resulted in an enzyme that bound mismatched substrates but was unable to catalyze base removal. Mutation of Met-269 in a motif with a postulated role in protein-substrate interaction selectively inactivated stable binding of the enzyme to mismatched substrates but not so its glycosylase activity. These results establish that the structure function model postulated for the E. coli enzyme is largely applicable to the human TDG. We further provide evidence for G.U being the preferred substrate of TDG, not only at the mismatch recognition step of the reaction but also in base hydrolysis, and for the importance of stable complementary strand interactions by TDG to compensate for its comparably poor hydrolytic potential.     

Classical and slow-binding inhibitors of human type II arginase

Arginases catalyze the hydrolysis of L-arginine to yield L-ornithine and urea. Recent studies indicate that arginases, both the type I and type II isozymes, participate in the regulation of nitric oxide production by modulating the availability of arginine for nitric oxide synthase. Due to the reciprocal regulation between arginase and nitric oxide synthase, arginase inhibitors have therapeutic potential in treating nitric oxide-dependent smooth muscle disorders, such as erectile dysfunction. We demonstrate the competitive inhibition of the mitochondrial human type II arginase by N(omega)-hydroxy-L-arginine, the intermediate in the reaction catalyzed by nitric oxide synthase, and its analogue N(omega)-hydroxy-nor-L-arginine, with K(i) values of 1.6 microM and 51 nM at pH 7.5, respectively. We also demonstrate the inhibition of human type II arginase by the boronic acid-based transition-state analogues 2(S)-amino-6-boronohexanoic acid (ABH) and S-(2-boronoethyl)-L-cysteine (BEC), which are known inhibitors of type I arginase. At pH 7.5, both ABH and BEC are classical, competitive inhibitors of human type II arginase with K(i) values of 0.25 and 0.31 microM, respectively. However, at pH 9.5, ABH and BEC are slow-binding inhibitors of the enzyme with K(i) values of 8.5 and 30 nM, respectively. The findings presented here indicate that the design of arginine analogues with uncharged, tetrahedral functional groups will lead to the development of more potent inhibitors of arginases at physiological pH.     

Mechanism of substrate recognition by drug-resistant human immunodeficiency virus type 1 protease variants revealed by a novel structural intermediate

Human immunodeficiency virus type 1 (HIV-1) protease processes and cleaves the Gag and Gag-Pol polyproteins, allowing viral maturation, and therefore is an important target for antiviral therapy. Ligand binding occurs when the flaps open, allowing access to the active site. This flexibility in flap geometry makes trapping and crystallizing structural intermediates in substrate binding challenging. In this study, we report two crystal structures of two HIV-1 protease variants bound with their corresponding nucleocapsid-p1 variant. One of the flaps in each of these structures exhibits an unusual "intermediate" conformation. Analysis of the flap-intermediate and flap-closed crystal structures reveals that the intermonomer flap movements may be asynchronous and that the flap which wraps over the P3 to P1 (P3-P1) residues of the substrate might close first. This is consistent with our hypothesis that the P3-P1 region is crucial for substrate recognition. The intermediate conformation is conserved in both the wild-type and drug-resistant variants. The structural differences between the variants are evident only when the flaps are closed. Thus, a plausible structural model for the adaptability of HIV-1 protease to recognize substrates in the presence of drug-resistant mutations has been proposed.     

The second-shell metal ligands of human arginase affect coordination of the nucleophile and substrate

The active sites of eukaryotic arginase enzymes are strictly conserved, especially the first- and second-shell ligands that coordinate the two divalent metal cations that generate a hydroxide molecule for nucleophilic attack on the guanidinium carbon of l-arginine and the subsequent production of urea and l-ornithine. Here by using comprehensive pairwise saturation mutagenesis of the first- and second-shell metal ligands in human arginase I, we demonstrate that several metal binding ligands are actually quite tolerant to amino acid substitutions. Of >2800 double mutants of first- and second-shell residues analyzed, we found more than 80 unique amino acid substitutions, of which four were in first-shell residues. Remarkably, certain second-shell mutations could modulate the binding of both the nucleophilic water/hydroxide molecule and substrate or product ligands, resulting in activity greater than that of the wild-type enzyme. The data presented here constitute the first comprehensive saturation mutagenesis analysis of a metallohydrolase active site and reveal that the strict conservation of the second-shell metal binding residues in eukaryotic arginases does not reflect kinetic optimization of the enzyme during the course of evolution.     

Mutational analysis of the human T-cell leukemia virus type I trans-acting rex gene product.

Expression of the human T-cell leukemia virus type I (HTLV-I) rex gene is a prerequisite for the expression of the retroviral structural proteins. We have generated internal deletion mutants of this 27-kDa nucleolar trans-acting gene product to define functional domains in the Rex protein. The phenotype of the various mutant proteins was tested on the homologous HTLV-I rex response element sequence and the heterologous human immunodeficiency virus type 1 (HIV-1) rev response element sequence. Our results indicate that a region between amino acid residues 55 and 132 in the 189-amino-acid Rex protein is required for Rex-mediated trans activation on both retroviral response element sequences. In addition, substitution of the Rex nuclear localization signal by a sequence of the HIV-1 rev gene product targets the Rex protein to the correct subcellular compartment required for Rex function.