Active Site Aspartic Acid Residues Research Articles

Escherichia coli DNA repair helicase Lhr is also a uracil-DNA glycosylase.

DNA glycosylases protect genetic fidelity during DNA replication by removing potentially mutagenic chemically damaged DNA bases. Bacterial Lhr proteins are well-characterized DNA repair helicases that are fused to additional 600-700 amino acids of unknown function, but with structural homology to SecB chaperones and AlkZ DNA glycosylases. Here, we identify that Escherichia coli Lhr is a uracil-DNA glycosylase (UDG) that depends on an active site aspartic acid residue. We show that the Lhr DNA helicase activity is functionally independent of the UDG activity, but that the helicase domains are required for fully active UDG activity. Consistent with UDG activity, deletion of lhr from the E. coli chromosome sensitized cells to oxidative stress that triggers cytosine deamination to uracil. The ability of Lhr to translocate single-stranded DNA and remove uracil bases suggests a surveillance role to seek and remove potentially mutagenic base changes during replication stress.

Structural and Hydrodynamic Characterization of Dimeric Human Oligoadenylate Synthetase 2

Oligoadenylate synthetases (OASs) are a family of interferon-inducible enzymes that require double-stranded RNA (dsRNA) as a cofactor. Upon binding dsRNA, OAS undergoes a conformational change and is activated to polymerize ATP into 2′-5′-oligoadenylate chains. The OAS family consists of several isozymes, with unique domain organizations to potentially interact with dsRNA of variable length, providing diversity in viral RNA recognition. In addition, oligomerization of OAS isozymes, potentially OAS1 and OAS2, is hypothesized to be important for 2′-5′-oligoadenylate chain building. In this study, we present the solution conformation of dimeric human OAS2 using an integrated approach involving small-angle x-ray scattering, analytical ultracentrifugation, and dynamic light scattering techniques. We also demonstrate OAS2 dimerization using immunoprecipitation approaches in human cells. Whereas mutation of a key active-site aspartic acid residue prevents OAS2 activity, a C-terminal mutation previously hypothesized to disrupt OAS self-association had only a minor effect on OAS2 activity. Finally, we also present the solution structure of OAS1 monomer and dimer, comparing their hydrodynamic properties with OAS2. In summary, our work presents the first, to our knowledge, dimeric structural models of OAS2 that enhance our understanding of the oligomerization and catalytic function of OAS enzymes.

Abstract LB-034: Identification of novel autophosphorylation structures in crystals of protein kinases

Abstract Cancer therapy depends heavily on the ability to effectively control the activity of oncogenic kinases. Autophosphorylation is a common regulatory mechanism of kinases in signaling pathways, and commonly elevated in cancer. Several autophosphorylation complexes have been identified from within crystals of protein kinases, with a known autophosphorylation site of one kinase monomer sitting in the active site of another monomer of the same protein in the crystal. We have utilized a structural bioinformatics method to identify all such autophosphorylation complexes in X-ray crystal structures in the Protein Data Bank by generating all unique kinase/kinase interfaces within and between asymmetric units of each crystal and measuring the distance between the hydroxyl oxygen of the autophosphorylation sites and the oxygen atoms of the active site aspartic acid residue side chain. With this approach, we have identified 15 autophosphorylation complexes in the PDB, of which 5 complexes have not previously been described. Of greatest interest are five structures of activation loop autophosphorylation - PAK1 (T423), IRAK4 (T345), IGF1R (Y1165 and Y1166), and LCK (Y394), two of which we have identified for the first time (IGF1R-Y1166 and LCK). We show that 269 human kinases have potential S/T or Y phosphorylation sites at positions analogous to the PAK1, IGF1R-Y1165/LCK, and IGF1R-Y1166 structures, and that there are 182 such positions that are annotated as phosphorylation sites in Uniprot. To assess the functional importance of the LCK dimer, we performed mutational analysis of residues in the autophosphorylation complex interface of LCK and found that mutations disrupting the interface either severely impaired autophosphorylation (T445D and N446D) or increased it (P447L,A,G). The P447L mutation has been previously found in a T-cell leukemia cell line and associated with activation of LCK. Three structures of receptor tyrosine kinases contain autophosphorylation complexes of the juxtamembrane segment just N-terminal to the kinase domain, two of which are identified for the first time. One of these, CSF1R (Y561) is a homologous site to a known c-KIT (Y568) autophosphorylation structure. The other is in EPHA2 (Y594), which is homologous to Y570 in c-KIT, which is also an autophosphorylation site. Twenty receptor tyrosine kinases contain autophosphorylation sites at one or both of these positions. Phosphorylation at these sites is associated with interaction with SRC family kinases and other downstream effectors of receptor tyrosine kinase signaling. These structures provide critical information on domain-domain interactions and substrate specificity in autophosphorylation, as well as opportunities for understanding the role of certain cancer driver mutations and the development of non-ATP-competitive inhibitors that block dimerization shown in these structures. Note: This abstract was not presented at the meeting. Citation Format: Qifang Xu, Kimberly Malecka, Jeffrey Peterson, Roland L. Dunbrack. Identification of novel autophosphorylation structures in crystals of protein kinases. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-034. doi:10.1158/1538-7445.AM2015-LB-034

The Handling of the Mechanistic Probe 5-Fluorouridine by the Pseudouridine Synthase TruA and Its Consistency with the Handling of the Same Probe by the Pseudouridine Synthases TruB and RluA

RNA containing 5-fluorouridine (F(5)U) had previously been used to examine the mechanism of the pseudouridine synthase TruA, formerly known as pseudouridine synthase I [Gu et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 14270-14275]. From that work, it was reasonably concluded that the pseudouridine synthases proceed via a mechanism involving a Michael addition by an active site aspartic acid residue to the pyrimidine ring of uridine or F(5)U. Those conclusions rested on the assumption that the hydrate of F(5)U was obtained after digestion of the product RNA and that hydration resulted from hydrolysis of the ester intermediate between the aspartic acid residue and F(5)U. As reported here, (18)O labeling definitively demonstrates that ester hydrolysis does not give rise to the observed hydrated product and that digestion generates not the expected mononucleoside product but rather a dinucleotide between a hydrated isomer of F(5)U and the following nucleoside in RNA. The discovery that digestion products are dinucleotides accounts for the previously puzzling differences in the isolated products obtained following the action of the pseudouridine synthases TruB and RluA on F(5)U in RNA.

Evidence for dimeric BACE-mediated APP processing

β-Secretase (BACE) is an aspartyl protease, which proteolytically processes amyloid precursor protein, making BACE an interesting pharmacological target in Alzheimer’s disease. To study the enzymatic function of BACE, we mutated either of the two aspartic acid residues in the active site of BACE. This rendered BACE functionally inactive without affecting the degree of glycosylation or endosomal localization. In contrast, substituting both active site aspartic acid residues produced a functionally inactive, endoplasmic reticulum-retained and partially glycosylated BACE. Interestingly, co-expression of the two single active site mutants partially restored β-site cleavage of amyloid precursor protein, and the restored activity was inhibited with similar dose-dependency and potency as wildtype BACE by a small molecule inhibitor raised against BACE. In sum, our data suggest that two different active site mutants can complement each other in a partially functional BACE dimer and mediate APP processing.

Novel peptide-based pepsin inhibitors containing an epoxide group

1,2-Epoxy-3-(p-nitrophenoxy)propane (EPNP) is known to inhibit pepsin A and other aspartic proteinases by reacting with the active site aspartic acid residue(s). However, the reaction is considerably slow in general, and therefore, it is desirable to develop similar reagents that are capable of inhibiting these enzymes more rapidly. In the present study, we synthesized a series of novel inhibitors which have a reactive epoxide group linked with peptide by a hydrazide bond, with a general structure: Iva-L-Val-L-Val-(L-AA)n-N2H2-ES-OEt (n = 0∼2) (Iva, isovaleryl; AA, bulky hydrophobic or aromatic amino acid residue; ES, epoxysuccinyl). These inhibitors were shown to inhibit porcine pepsin A remarkably faster than EPNP.

A New Perspective on Response Regulator Activation

Two-component signaling pathways are the most prevalent of strategies used by bacteria for coupling environmental signals to adaptive responses (10). The response regulator component typically functions at the end of the pathway, with the conserved regulatory/receiver domain acting as a phosphorylation-activated switch to control an associated or attached effector domain that elicits the output response (24). In the simplest mechanistic model, these domains are considered to exist in two conformations, an inactive (“off”) state and an active (“on”) state, with phosphorylation at a conserved active-site aspartic acid residue serving to shift the equilibrium toward the active state (22). In this issue of the Journal of Bacteriology, Dyer and Dahlquist provide direct structural evidence that this model is overly simplistic (8). Their crystallographic characterization of the unphosphorylated Escherichia coli chemotaxis response regulator CheY bound to a peptide of its effector protein target, the flagellar switch protein FliM, reveals a conformation that is intermediate between inactive and active states. This finding provides evidence against a strict two-state mechanism in which the orientations of key residues that distinguish the inactive and active states are presumed to be obligatorily coupled. The intermediate state reported in their study not only provides insight into the mechanism of switching between inactive and active states but also has potential physiological relevance for some response regulators.

Active-site residues in the type IV prepilin peptidase homologue PibD from the archaeon Sulfolobus solfataricus.

Archaeal preflagellin peptidases and bacterial type IV prepilin peptidases belong to a family of aspartic acid proteases that cleave the leader peptides of precursor proteins with type IV prepilin signal sequences. The substrate repertoire of PibD from the crenarchaeon Sulfolobus solfataricus is unusually diverse. In addition to flagellin, PibD cleaves three sugar-binding proteins unique to this species and a number of proteins with unknown function. Here we demonstrate that PibD contains two aspartic acid residues that are essential for cleavage activity. An additional pair of aspartic acids in a large cytoplasmic loop is also important for function and is possibly involved in leader peptide recognition. Combining the results of transmembrane segment predictions and cysteine-labeling experiments, we suggest a membrane topology model for PibD with the active-site aspartic acid residues exposed to the cytosol.

A replacement of the active-site aspartic acid residue 293 in mouse cathepsin D affects its intracellular stability, processing and transport in HEK-293 cells.

The substitution of an active-site aspartic acid residue by asparagine in the lysosomal protease cathepsin D (CTSD) results in a loss of enzyme activity and severe cerebrocortical atrophy in a novel form of neuronal ceroid lipofuscinosis in sheep [Tyynelä, Sohar, Sleat, Gin, Donnelly, Baumann, Haltia and Lobel (2000) EMBO J. 19, 2786-2792]. In the present study we have introduced the corresponding mutation by replacing aspartic acid residue 293 with asparagine (D293N) into the mouse CTSD cDNA to analyse its effect on synthesis, transport and stability in transfected HEK-293 cells. The complete inactivation of mutant D293N mouse CTSD was confirmed by a newly developed fluorimetric quantification system. Moreover, in the heterologous overexpression systems used, mutant D293N mouse CTSD was apparently unstable and proteolytically modified during early steps of the secretory pathway, resulting in a loss of mass by about 1 kDa. In the affected sheep, the endogenous mutant enzyme was stable but also showed the shift in its molecular mass. In HEK-293 cells, the transport of the mutant D293N mouse CTSD to the lysosome was delayed and associated with a low secretion rate compared with wild-type CTSD. These data suggest that the mutation may result in a conformational change which affects stability, processing and transport of the enzyme.

Stabilization of Charges and Protonation States in the Active Site of the Protein Tyrosine Phosphatases: A Computational Study

The initial step of the dephosphorylation of phosphotyrosine by the protein tyrosine phosphatases (PTPases) requires an active-site cysteine residue to be in its deprotonated (thiolate) form so as to make a nucleophilic attack on the phosphate to form a phosphoenzyme intermediate. At the same time, an active-site aspartic acid residue must be in its protonated form to serve as a general acid donating a proton to the leaving tyrosyl moiety. The protein must therefore provide an unusual electrostatic environment in the active site to maintain these protonation states in the free enzyme and in the Michaelis complex with the substrate, whose phosphate group is in the −2 charged form. We present electrostatic calculations for the pKa of the ionizable side chains of four different PTPases. The study includes Michaelis complexes with the substrate, as well as free enzymes. The calculations consistently predict that in the neutral to mildly acidic pH range, the active-site cysteine residue is deprotonated and the...

Aspartic Proteases from the Nematode Caenorhabditis elegans: STRUCTURAL ORGANIZATION AND DEVELOPMENTAL AND CELL-SPECIFIC EXPRESSION OF asp-1

A Caenorhabditis elegans gene (asp-1) and cDNA that encode a homologue of cathepsin D aspartic protease were cloned and characterized. The asp-1 mRNA is transcribed from a single exon, and it begins with the SL1 trans-splice leader sequence. The protein (ASP-1) is expressed as a 396-amino acid, 42.7-kDa pre-pro-peptide that is post-translationally processed into a approximately 40-kDa lysosomal protein. ASP-1 shares approximately 60% sequence identity with the aspartic protease precursor from the nematode Strongyloides stercoralis. The amino acid sequences adjacent to the two active site aspartic acid residues in ASP-1 are 100% identical to those in other eukaryotic aspartic proteases. In addition, ASP-1 contains conserved, potential disulfide bond-forming cysteine residues and N-glycosylation sites. The asp-1 gene is exclusively transcribed in the intestinal cells, with the highest levels of expression observed at late embryonic and early larval stages of development. asp-1 transcription is not observed in adult nematodes or mature larvae. Furthermore, transcription predominantly occurs in eight anterior cells of the intestine (int6-int8). Analyses of ASP-1 nucleotide and amino acid sequences revealed the presence of five additional C. elegans aspartic proteases.

Structure based prediction of binding affinity of human immunodeficiency virus-1 protease inhibitors.

A series of computations were performed to derive a strategy for the prediction of binding affinities of non-peptidic human immunodeficiency virus-1 (HIV-1) protease inhibitors. This paper describes the development of a 3D quantitative structure-activity relationship (3D-QSAR) methodology by using receptor information of HIV-1 protease. The docking and molecular dynamics simulations were performed on a model ligand/enzyme complex to optimize the variables involved in the generation of ligand/enzyme models. The protonation scheme of the active site aspartic acid residues of HIV-1 protease was derived from a computational study. The active site aspartate is monoprotonated with a proton placed on the OD1 atom of the ASP B25. This protocol of docking and molecular dynamics (MD) simulation was then used to derive the ligand-enzyme complexes of the molecules used in the present study. The molecular mechanics interaction descriptors were calculated from these ligand/enzyme models. A partial least squares (PLS) method was used to derive a linear correlation between the interaction descriptors and the biological activity. A good correlation was observed when the change in the energy of the ligand upon complex formation and the electrostatic contributions to the solvation energy of the ligand were included in the QSAR analysis. A highest cross-validated q2 value of 0.649 was observed. This model had a conventional r2 of 0.725, and when this model was used to predict the activity of the external test set, it produced a r2pred of 0.761. The total interaction energy was partitioned into interactions in different subsites and interactions with each of the amino acid residues of the enzyme. The PLS analysis using these descriptors helped to identify the important interactions which can be exploited for the design of HIV-1 protease inhibitors.

Dissection of the pH dependence of inhibitor binding energetics for an aspartic protease: direct measurement of the protonation states of the catalytic aspartic acid residues.

The catalytic activity and inhibitor binding energetics of enzymes are often pH-dependent properties. Aspartic proteases comprise an important class of enzyme targets for structure-based drug design. We have performed a complete thermodynamic study of pepstatin binding to plasmepsin II, an aspartic proteinase found in Plasmodium falciparum, using isothermal titration calorimetry and circular dichroism. Thermodynamic parameters (DeltaG, DeltaH, DeltaCp, and DeltaS) were measured as functions of both pH and temperature. In the pH range from 4.5 to 7.0, pepstatin binding is accompanied by proton transfer between the solvent and the complex. We used thermodynamic proton linkage theory to derive both the pH-independent binding energetics for pepstatin and the number and pKa values of ionizable residues whose pKa values change during ligand binding. These residues were identified as the two catalytic aspartates, with pKas of 6.5 and 3.0, and His 164, with a pKa of 7.5, based on the three-dimensional structure of the pepstatin-plasmepsin II complex. At pH 5.0, where the protease has optimum activity, the proton transfer process contributes almost 40% of the total binding free energy change and the total charge of the active-site aspartic acid residues is -1. These experimental results provide direct measurement for the protonation states of the catalytic aspartates in the presence of bound ligands. Comparison of the thermodynamic and structural data for pepstatin binding with human cathepsin D, a lysosomal aspartic protease that shares 35% sequence identity with plasmepsin II, suggests that the energetic differences between these two proteins are due to a higher interdomain flexibility in plasmepsin II.

Role of the active-site carboxylate in dihydrofolate reductase: kinetic and spectroscopic studies of the aspartate 26-->asparagine mutant of the Lactobacillus casei enzyme.

A mutant of Lactobacillus casei dihydrofolate reductase, D26N, in which the active site aspartic acid residue has been replaced by asparagine by oligonucleotide-directed mutagenesis has been studied by NMR and optical spectroscopy and its kinetic behavior characterized in detail. On the basis of comparisons of a large number of chemical shifts and NOEs, it is clear that there are only very slight structural differences between the methotrexate complexes of the wild-type and mutant enzymes and that these are restricted to the immediate environment of the substitution. The data suggest a slight difference in orientation of the pteridine ring in the binding site in the mutant enzyme. Both NMR and UV spectroscopy show that methotrexate is protonated on N1 when bound to the wild-type enzyme but not when bound to the mutant. Binding constant measurements by fluorescence quenching and steady-state kinetic measurements of dihydrofolate (FH2) and folate reduction show that the substitution has little or no effect on substrate, coenzyme, and inhibitor binding (< 7-fold increase in Kd) and only a modest effect on kcat (up to a factor of 9 for FH2 and 25 for folate) and kcat/KM (up to a factor of 13 for FH2 and 14 for folate). Measurements of deuterium isotope effects and direct measurements of hydride ion transfer and product release by stopped-flow methods revealed that for the mutant enzyme hydride ion transfer is rate-limiting across the pH range 5-8. This allowed a direct comparison of the rate of hydride ion transfer in the wild-type and mutant enzymes; the asparagine substitution was found to decrease this rate by 62-fold at pH 5.5 and 9-fold at pH 7.5. This effect is much smaller than that seen for the corresponding mutant of Escherichia coli dihydrofolate reductase [Howell, E. E., Villafranca, J. E., Warren, M. S., Oatley, S. J., & Kraut, J. (1986) Science 231, 1123-1128], estimated as a 1000-fold decrease in the rate of hydride ion transfer. The change in pH dependence of kcat resulting from the substitution is consistent with, but does not prove, the idea that the group of pK 6.0 which must be protonated for hydride ion transfer to occur is Asp26. For folate reduction, the pH dependence of kcat is determined by two pKs, one of which, pK 5, disappears in the mutant enzyme, suggesting that it may correspond to ionization of Asp26.(ABSTRACT TRUNCATED AT 400 WORDS)

Constitutive mutations of Agrobacterium tumefaciens transcriptional activator virG.

The virulence (vir) genes of Agrobacterium tumefaciens Ti plasmids are positively regulated by virG in conjunction with virA and plant-derived inducing molecules. A procedure that utilizes both genetic selection and a genetic screen was developed to isolate mutations in virG that led to elevated levels of vir gene expression in the absence of virA and plant phenolic inducers. Mutants were isolated at a frequency of 1 in 10(7) to 10(8). Substitution mutations at two positions in the virG coding region were found to result in the desired phenotype. One mutant had an asparagine-to-aspartic acid substitution at residue 54, and the other contained an isoleucine-to-leucine substitution at residue 106. In both cases, the mutant phenotype required the presence of the active-site aspartic acid residue at position 52. Further analysis showed that no other substitution at residue 54 resulted in a constitutive phenotype. In contrast, several substitutions at residue 106 led to a constitutive phenotype. The possible roles of the residues at positions 54 and 106 in VirG function are discussed.

Conversion of citrate synthase into citryl-CoA lyase as a result of mutation of the active-site aspartic acid residue to glutamic acid

The active-site aspartic acid residue, Asp-362, of Escherichia coli citrate synthase was changed by site-directed mutagenesis to Glu-362, Asn-362 or Gly-362. Only very low catalytic activity could be detected with the Asp----Asn and Asp----Gly mutations. The Asp----Glu mutation produced an enzyme that expressed about 0.8% of the overall catalytic rate, and the hydrolysis step in the reaction, monitored as citryl-CoA hydrolysis, was inhibited to a similar extent. However, the condensation reaction, measured in the reverse direction as citryl-CoA cleavage to oxaloacetate and acetyl-CoA, was not affected by the mutation, and this citryl-CoA lyase activity was the major catalytic activity of the mutant enzyme. This high condensation activity in an enzyme in which the subsequent hydrolysis step was about 98% inhibited permitted considerable exchange of the methyl protons of acetyl-CoA during catalysis by the mutant enzyme. The Km for oxaloacetate was not significantly altered in the D362E mutant enzyme, whereas the Km for acetyl-CoA was about 5 times lower. A mechanism is proposed in which Asp-362 is involved in the hydrolysis reaction of this enzyme, and not as a base in the deprotonation of acetyl-CoA as recently suggested by others. [Karpusas, Branchaud & Remington (1990) Biochemistry 29, 2213-2219; Alter, Casazza, Zhi, Nemeth, Srere & Evans, (1990) Biochemistry 29, 7557-7563].

Inhibition of Scytalidium lignicolum acid protease B by 1-diazo-3-phenyl-2-propanone.

The acid protease B from Scytalidium lignicolum, a pepstatin-insensitive acid protease, was modified with l-diazo-3-phenyl-2-propanone (DPP) in the presence of cupric ions. About 1 mol of DPP per mol of enzyme was incorporated with a concomitant loss of the activity. The modified enzyme was digested with thermolysin and DPP-labeled peptides were separated. One peptide, called SI, was purified and its amino acid sequence was found to be Ala-Thr-Ser-Asp-Thr-Ser-Gly-Ser, which corresponds to the sequence of residue Nos. 95 ~ 102 of the enzyme. Alkali treatment of the modified peptide resulted in complete removal of the modifier, suggesting that DPP is esterlinked to Asp-98 of the enzyme. The amino acid sequence around Asp-98 of the enzyme was significantly homologous to those around the active site aspartic acid residues of penicillopepsin and porcine pepsin. These results suggest that Asp 98 is one of the active site amino acids of the enzyme.

Cloning and sequence analysis of cDNA for Irpex lacteus aspartic proteinase.

To find the primary sequence of Irpex lacteus aspartic proteinase (ILAP), a cDNA library of J. lacteus mRNA in pBR322 was constructed. A clone, which had an insert size of about 1.2 kilobase pairs, was found to contain the coding region of the mature enzyme. The deduced amino acid sequence showed that the enzyme consisted of 340 amino acid residues with a molecular weight of 35,000. Cysteine and methionine were not found in the enzyme and two putative /V-glycosylation sites were indicated. The lack of S-S bridges in the molecule is a striking feature of the enzyme. The alignment of the sequence of the enzyme against other aspartic proteinases revealed homology around the active site aspartic acid residues.

SECONDARY STRUCTURE OF SOME ASPARTYL PROTEINASES

A structural study of some aspartyl proteinases was undertaken. Secondary structure prediction methods indicate that chymosin, pepsin, penicillopepsin and Mucor miehei proteinase have relatively high proportions of β-sheet with active site aspartic acid residues located in β-turn regions. Secondary structure determination from far-UV CD spectral data support the above finding that the aspartyl proteinases have a high proportion of β-sheet. The proportion of β-sheet generally decreased at pH values greater than 6.3. More extensive unfolding occurred with pepsin and penicillopepsin than chymosin, Mucor miehei proteinase, Mucor pusillus proteinase and Endothia parasitica proteinase in the neutral to alkaline pH range. Results obtained from the near-UV CD spectra of the aspartyl proteinases indicate a change in spectra in the neutral to alkaline pH range which suggests the importance of aromatic groups to tertiary structure stability.