Mutational analysis of duck delta 2 crystallin and the structure of an inactive mutant with bound substrate provide insight into the enzymatic mechanism of argininosuccinate lyase.

The major soluble avian eye lens protein, delta crystallin, is highly homologous to the housekeeping enzyme argininosuccinate lyase (ASL). ASL is part of the urea and arginine-citrulline cycles and catalyzes the reversible breakdown of argininosuccinate to arginine and fumarate. In duck lenses, there are two delta crystallin isoforms that are 94% identical in amino acid sequence. Only the delta2 isoform has maintained ASL activity and has been used to investigate the enzymatic mechanism of ASL. The role of the active site residues Ser-29, Asp-33, Asp-89, Asn-116, Thr-161, His-162, Arg-238, Thr-281, Ser-283, Asn-291, Asp-293, Glu-296, Lys-325, Asp-330, and Lys-331 have been investigated by site-directed mutagenesis, and the structure of the inactive duck delta2 crystallin (ddeltac2) mutant S283A with bound argininosuccinate was determined at 1.96 A resolution. The S283A mutation does not interfere with substrate binding, because the 280's loop (residues 270-290) is in the open conformation and Ala-283 is more than 7 A from the substrate. The substrate is bound in a different conformation to that observed previously indicating a large degree of conformational flexibility in the fumarate moiety when the 280's loop is in the open conformation. The structure of the S283A ddeltac2 mutant and mutagenesis results reveal that a complex network of interactions of both protein residues and water molecules are involved in substrate binding and specificity. Small changes even to residues not involved directly in anchoring the argininosuccinate have a significant effect on catalysis. The results suggest that either His-162 or Thr-161 are responsible for proton abstraction and reinforce the putative role of Ser-283 as the catalytic acid, although we cannot eliminate the possibility that arginine is released in an uncharged form, with the solvent providing the required proton. A detailed enzymatic mechanism of ASL/ddeltac2 is presented.     

Mechanisms for intragenic complementation at the human argininosuccinate lyase locus.

Argininosuccinate lyase (ASL) is a homotetrameric enzyme that catalyzes the reversible cleavage of argininosuccinate to arginine and fumarate. Deficiencies in the enzyme result in the autosomal, recessive disorder argininosuccinic aciduria. Considerable clinical and genetic heterogeneity is associated with this disorder, which is thought to be a consequence of the extensive intragenic complementation identified in patient strains. Our ability to predict genotype-phenotype relationships is hampered by the current lack of understanding of the mechanisms by which complementation can occur. The 3-dimensional structure of wild-type ASL has enabled us to propose that the complementation between two ASL active site mutant subunits, Q286R and D87G, occurs through a regeneration of functional active sites in the heteromutant protein. We have reconstructed this complementation event, both in vivo and in vitro, using recombinant proteins and have confirmed this hypothesis. The complementation events between Q286R and two nonactive site mutants, M360T and A398D, have also been characterized. The M360T and A398D substitutions have adverse effects on the thermodynamic stability of the protein. Complementation between either the M360T or the A398D mutant and the stable Q286R mutant occurs through the formation of a more stable heteromeric protein with partial recovery of catalytic activity. The detection and characterization of a novel complementation event between the A398D and D87G mutants has shown how complementation in patients with argininosuccinic aciduria may correlate with the clinical phenotype.     

A duck delta1 crystallin double loop mutant provides insight into residues important for argininosuccinate lyase activity

Delta-crystallin is directly related to argininosuccinate lyase (ASL), and catalyzes the reversible hydrolysis of argininosuccinate to arginine and fumarate. Two delta-crystallin isoforms exist in duck lenses, delta1 and delta2, which are 94% identical in amino acid sequence. Although the sequences of duck delta2-crystallin (ddeltac2) and duck delta1-crystallin (ddeltac1) are 69 and 71% identical to that of human ASL, respectively, only ddeltac2 has maintained ASL activity. Domain exchange experiments and comparisons of various delta-crystallin structures have suggested that the amino acid substitutions in the 20's (residues 22-31) and 70's (residues 74-89) loops of ddeltac1 are responsible for the loss of enzyme activity in this isoform. To test this hypothesis, a double loop mutant (DLM) of ddeltac1 was constructed in which all the residues that differ between the two isoforms in the 20's and 70's loops were mutated to those of ddeltac2. Contrary to expectations, kinetic analysis of the DLM found that it was enzymatically inactive. Furthermore, binding of argininosuccinate by the DLM, as well as the ddeltac1, could not be detected by isothermal titration calorimetry (ITC). To examine the conformation of the 20's and 70's loops in the DLM, and to understand why the DLM is unable to bind the substrate, its structure was determined to 2.5 A resolution. Comparison of this structure with both wild-type ddeltac1 and ddeltac2 structures reveals that the conformations of the 20's and 70's loops in the DLM mutant are very similar to those of ddeltac2. This suggests that the five amino acid substitutions in domain 1 which lie outside of the two loop regions and which are different in the DLM, and ddeltac2, must be important enzymatically. The structure of the DLM in complex with sulfate was also determined to 2.2 A resolution. This structure demonstrates that the conformational changes of the 280's loop and domain 3, previously observed in ddeltac1, also occur in the DLM upon sulfate binding, reinforcing the hypothesis that these events may occur in the active ddeltac2 protein during catalysis.     

Three subunits contribute amino acids to the active site of tetrameric adenylosuccinate lyase: Lys268 and Glu275 are required

Tetrameric adenylosuccinate lyase (ASL) of Bacillus subtilis catalyzes the cleavage of adenylosuccinate to form AMP and fumarate. We previously reported that two distinct subunits contribute residues to each active site, including the His68 and His89 from one and His141 from a second subunit [Brosius, J. L., and Colman, R. F. (2000) Biochemistry 39, 13336-13343]. Glu(275) is 2.8 A from His141 in the ASL crystal structure, and Lys268 is also in the active site region; Glu275 and Lys268 come from a third, distinct subunit. Using site-directed mutagenesis, we have replaced Lys268 by Arg, Gln, Glu, and Ala, with specific activities of the purified mutant enzymes being 0.055, 0.00069, 0.00028, and 0.0, respectively, compared to 1.56 units/mg for wild-type (WT) enzyme. Glu275 was substituted by Gln, Asp, Ala, and Arg; none of these homogeneous mutant enzymes has detectable activity. Circular dichroism and light scattering reveal that neither the secondary structure nor the oligomeric state of the Lys268 mutant enzymes has been perturbed. Native gel electrophoresis and circular dichroism indicate that the Glu275 mutant enzymes are tetramers, but their conformation is altered slightly. For K268R, the K(m)s for all substrates are similar to WT enzyme. Binding studies using [2-3H]-adenylosuccinate reveal that none of the Glu275 mutant enzymes, nor inactive K268A, can bind substrate. We propose that Lys268 participates in binding substrate and that Glu275 is essential for catalysis because of its interaction with His141. Incubation of H89Q with K268Q or E275Q leads to restoration of up to 16% WT activity, while incubation of H141Q with K268Q or E275Q results in 6% WT activity. These complementation studies provide the first functional evidence that a third subunit contributes residues to each intersubunit active site of ASL. Thus, adenylosuccinate lyase has four active sites per enzyme tetramer, each of which is formed from regions of three subunits.     

Structural studies of duck delta 1 and delta 2 crystallin suggest conformational changes occur during catalysis

Duck delta1 and delta2 crystallin are 94% identical in amino acid sequence, and while delta2 crystallin is the duck orthologue of argininosuccinate lyase (ASL) and catalyzes the reversible breakdown of argininosuccinate to arginine and fumarate, the delta1 isoform is enzymatically inactive. The crystal structures of wild type duck delta1 and delta2 crystallin have been solved at 2.2 and 2.3 A resolution, respectively, and the refinement of the turkey delta1 crystallin has been completed. These structures have been compared with two mutant duck delta2 crystallin structures. Conformational changes were observed in two regions of the N-terminal domain with intraspecies differences between the active and inactive isoforms localized to residues 23-32 and both intra- and interspecies differences localized to the loop of residues 74-89. As the residues implicated in the catalytic mechanism of delta2/ASL are all conserved in delta1, the amino acid substitutions in these two regions are hypothesized to be critical for substrate binding. A sulfate anion was found in the active site of duck delta1 crystallin. This anion, which appears to mimic the fumarate moiety of the argininosuccinate substrate, induces a rigid body movement in domain 3 and a conformational change in the loop of residues 280-290, which together would sequester the substrate from the solvent. The duck delta1 crystallin structure suggests that Ser 281, a residue strictly conserved in all members of the superfamily, could be the catalytic acid in the delta2 crystallin/ASL enzymatic mechanism.     

Recovery of argininosuccinate lyase activity in duck delta1 crystallin

Delta-crystallin, the major soluble protein component in the avian eye lens, is homologous to argininosuccinate lyase (ASL). Two delta-crystallin isoforms exist in ducks, delta1- and delta2-crystallin, which are 94% identical in amino acid sequence. While duck delta2-crystallin (ddeltac2) has maintained ASL activity, evolution has rendered duck delta1-crystallin (ddeltac1) enzymatically inactive. Previous attempts to regenerate ASL activity in ddeltac1 by mutating the residues in the 20s (residues 22-31) and 70s (residues 74-89) loops to those found in ddeltac2 resulted in a double loop mutant (DLM) which was enzymatically inactive (Tsai, M. et al. (2004) Biochemistry 43, 11672-82). This result suggested that one or more of the remaining five amino acid substitutions in domain 1 of the DLM contributes to the loss of ASL activity in ddeltac1. In the current study, residues Met-9, Val-14, Ala-41, Ile-43, and Glu-115 were targeted for mutagenesis, either alone or in combination, to the residues found in ddeltac2. ASL activity was recovered in the DLM by changing Met-9 to Trp, and this activity is further potentiated in the DLM-M9W mutant when Glu-115 is changed to Asp. The roles of Trp-9 and Asp-115 were further investigated by site-directed mutagenesis in wild-type ddeltac2. Changing the identity of either Trp-9 or Asp-115 in ddeltac2 resulted in a dramatic drop in enzymatic activity. The loss of activity in Trp-9 mutants indicates a preference for an aromatic residue at this position. Truncation mutants of ddeltac2 in which the first 8, 9, or 14 N-terminal residues were removed displayed either decreased or no ASL activity, suggesting residues 1-14 are crucial for enzymatic activity in ddeltac2. Our kinetic studies combined with available structural data suggest that the N-terminal arm in ASL/delta2-crystallin is involved in stabilizing regions of the protein involved in substrate binding and catalysis, and in completely sequestering the substrate from the solvent.     

Mutational Analysis of Bacterial NAD^＋-dependent DNA Ligase： Role of Motif IV in Ligation Catalysis

The bacterial DNA ligase as a multiple domain protein is involved in DNA replication, repair and recombination. Its catalysis of ligation can be divided into three steps. To delineate the roles of amino acid residues in motif IV in ligation catalysis, site-directed mutants were constructed in a bacterial NAD^＋-dependent DNA ligase from Thermus sp. TAK16D. It was shown that four conserved residues （D286, G287, V289 and K291） in motif IV had significant roles on the overall ligation. Under single turnover conditions, the observed apparent rates of D286E, G287A, V289I and K291R mutants were clearly reduced compared with that of WT ligase on both match and mismatch nicked substrates. The effects of D286E mutation on overall ligation may not only be ascribed to the third step. The G287A mutation has a major effect on the second step. The effects of V289I and K291R mutation on overall ligation are not on the third step, perhaps other aspects, such as conformation change of ligase protein in ligation catalysis, are involved. Moreover, the amino acid substitutions of above four residues were more sensitive on mismatch nicked substrate, indicating an enhanced ligation fidelity.     

Solution structure of the catalytic domain of gammadelta resolvase. Implications for the mechanism of catalysis

The site-specific DNA recombinase, gammadelta resolvase, from Escherichia coli catalyzes recombination of res site-containing plasmid DNA to two catenated circular DNA products. The catalytic domain (residues 1-105), lacking a C-terminal dimerization interface, has been constructed and the NMR solution structure of the monomer determined. The RMSD of the NMR conformers for residues 2-92 excluding residues 37-45 and 64-73 is 0.41 A for backbone atoms and 0.88 A for all heavy atoms. The NMR solution structure of the monomeric catalytic domain (residues 1-105) was found to be formed by a four-stranded parallel beta-sheet surrounded by three helices. The catalytic domain (residues 1-105), deficient in the C-terminal dimerization domain, was monomeric at high salt concentration, but displayed unexpected dimerization at lower ionic strength. The unique solution dimerization interface at low ionic strength was mapped by NMR. With respect to previous crystal structures of the dimeric catalytic domain (residues 1-140), differences in the average conformation of active-site residues were found at loop 1 containing the catalytic S10 nucleophile, the beta1 strand containing R8, and at loop 3 containing D67, R68 and R71, which are required for catalysis. The active-site loops display high-frequency and conformational backbone dynamics and are less well defined than the secondary structures. In the solution structure, the D67 side-chain is proximal to the S10 side-chain making the D67 carboxylate group a candidate for activation of S10 through general base catalysis. Four conserved Arg residues can function in the activation of the phosphodiester for nucleophilic attack by the S10 hydroxyl group. A mechanism for covalent catalysis by this class of recombinases is proposed that may be related to dimer interface dissociation.     

Protein-protein interactions in actin-myosin binding and structural effects of R405Q mutation: a molecular dynamics study

Detailed residue-wise interactions involved in the binding of myosin to actin in the rigor conformation without nucleotides have been examined using molecular dynamics simulations of the chicken skeletal myosin head complexed with two actin monomers, based on the cryo-microscopic model of Holmes et al. (Nature 2003;425:423-427). The overall interaction is largely electrostatic in nature, because of the charged residues in the four loops surrounding the central primary binding site. The 50k/20k loop, disordered in crystal structures and in simulations of free myosin in solution, was found to be in a conformation stabilized with 1 - 2 internal salt bridges. The cardiomyopathy loop forms 2 - 3 interprotein salt bridges with actin monomers upon binding, whereas its Arg405 residue, the mutation site associated with the hypertrophic cardiomyopathy, forms a strong salt bridge with Glu605 in the neighboring helix away from actin in the actin-bound myosin. The myopathy loop of the R405Q mutant maintains a high degree of two-strand beta-sheet character when bound to actin with the corresponding salt bridges broken.     

Disruption of a salt bridge dramatically accelerates subunit exchange in duck delta2 crystallin

Intragenic complementation is a unique property of oligomeric enzymes with which to study subunit-subunit interactions. Complementation occurs when different subunits, each possessing distinct mutations that render the individual homomutant proteins inactive, interact to form a heteromutant protein with partial recovery of activity. In this paper, complementation events between human argininosuccinate lyase (ASL) and its homolog, duck delta2 crystallin, were characterized. Different active site mutants in delta2 crystallin complement by the regeneration of native-like active sites as reported previously for ASL. The complementarity of the ASL and delta2 crystallin subunit interfaces was illustrated by the in vivo formation of active hybrid tetramers from inactive ASL and inactive delta2 crystallin mutants. Subunits of both ASL and delta2 crystallin do not dissociate and reassociate in vitro at room temperature, even after 6 days of incubation, indicating that the multimerization interface is very strong. However, disruption of a salt bridge network in the tetrameric interface of delta2 crystallin caused a drastic acceleration of subunit dissociation. Double mutants combining these interface mutants with active site mutants of delta2 crystallin were able to dissociate and reassociate to form active tetramers in vitro within hours. These results suggest that exchange of subunits may occur without unfolding of the monomer. Intragenic complementation in these interface mutants occurs by reintroducing the native salt bridge interaction upon hetero-oligomerization. Our studies demonstrate the value of intragenic complementation as a tool for investigating subunit-subunit interactions in oligomeric proteins.     

Crystal structure of an inactive duck delta II crystallin mutant with bound argininosuccinate

Delta-crystallin, the major soluble protein component of avian and reptilian eye lenses, is highly homologous to the urea cycle enzyme, argininosuccinate lyase (ASL). In duck lenses, there are two highly homologous delta crystallins, delta I and delta II, that are 94% identical in amino acid sequence. While delta II crystallin has been shown to exhibit ASL activity in vitro, delta I is enzymatically inactive. The X-ray structure of a His to Asn mutant of duck delta II crystallin (H162N) with bound argininosuccinate has been determined to 2.3 A resolution using the molecular replacement technique. The overall fold of the protein is similar to other members of the superfamily to which this protein belongs, with the active site located in a cleft formed by three different monomers in the tetramer. The active site of the H162N mutant structure reveals that the side chain of Glu 296 has a different orientation relative to the homologous residue in the H91N mutant structure [Abu-Abed et al. (1997) Biochemistry 36, 14012-14022]. This shift results in the loss of the hydrogen bond between His 162 and Glu 296 seen in the H91N and turkey delta I crystallin structures; this H-bond is believed to be crucial for the catalytic mechanism of ASL/delta II crystallin. Argininosuccinate was found to be bound to residues in each of the three monomers that form the active site. The fumarate moiety is oriented toward active site residues His 162 and Glu 296 and other residues that are part of two of the three highly conserved regions of amino acid sequence in the superfamily, while the arginine moiety of the substrate is oriented toward residues which belong to either domain 1 or domain 2. The analysis of the structure reveals that significant conformational changes occur on substrate binding. The comparison of this structure with the inactive turkey delta I crystallin reveals that the conformation of domain 1 is crucial for substrate affinity and that the delta I protein is almost certainly inactive because it can no longer bind the substrate.     

Role of charged amino acids conserved in the vasoactive intestinal polypeptide/secretin family of receptors on the secretin receptor functionality

The secretin receptor is a member of a large family of G-protein-coupled receptors that recognize polypeptide hormone and/or neuropeptides. Charged, conserved residues might play a key role in their function, either by interacting with the ligand or by stabilizing the receptor structure. Of the four charged amino acids that are conserved in the whole secretin receptor family, D49 and R83 (in the N-terminal domain) were probably important for the secretin receptor structure: replacement of D49 by H or R and of R83 by D severely reduced both the maximal response to secretin and its potency. No functional secretin receptor could be detected after replacement of R83 by L. Mutation of D49 to E, A, or N had no effect or reduced 5-fold the potency of secretin. The highly conserved positive charges found at the extracellular ends of TM III (K194) and IV (R255) were important for the secretin receptor function, as K194 mutation to A or Q and R255 mutation to Q or D decreased the secretin's affinity 15- to 1000-fold, respectively. Six extracellular charged residues are conserved in closely related receptors but not in the whole family. K121 (TM I) and R277 (TM V) were not important for functional secretin receptor expression. D174 (TM II) was necessary to stabilize the active receptor structure: the D174N mutant receptors were unable to stimulate normally the adenylate cyclase in response to secretin, and functional D174A receptors could not be found. Mutation of R255, E259 (second extracellular loop), and E351 (third extracellular loop) to uncharged residues reduced only 10- to 100-fold the secretin potency without changing its efficacy: these residues either stabilized the active receptor conformation or formed hydrogen rather than ionic bonds with secretin. Mutation of K121 (TM I) to Q or L and of R277 (TM V) to E or Q did not affect the receptor functional properties.     

Kinetic, stereochemical, and structural effects of mutations of the active site arginine residues in 4-oxalocrotonate tautomerase.

Three arginine residues (Arg-11, Arg-39, Arg-61) are found at the active site of 4-oxalocrotonate tautomerase in the X-ray structure of the affinity-labeled enzyme [Taylor, A. B., Czerwinski, R. M., Johnson, R. M., Jr., Whitman, C. P., and Hackert, M. L. (1998) Biochemistry 37, 14692-14700]. The catalytic roles of these arginines were examined by mutagenesis, kinetic, and heteronuclear NMR studies. With a 1,6-dicarboxylate substrate (2-hydroxymuconate), the R61A mutation showed no kinetic effects, while the R11A mutation decreased k(cat) 88-fold and increased K(m) 8.6-fold, suggesting both binding and catalytic roles for Arg-11. With a 1-monocarboxylate substrate (2-hydroxy-2,4-pentadienoate), no kinetic effects of the R11A mutation were found, indicating that Arg-11 interacts with the 6-carboxylate of the substrate. The stereoselectivity of the R11A-catalyzed protonation at C-5 of the dicarboxylate substrate decreased, while the stereoselectivity of protonation at C-3 of the monocarboxylate substrate increased in comparison with wild-type 4-OT, indicating the importance of Arg-11 in properly orienting the dicarboxylate substrate by interacting with the charged 6-carboxylate group. With 2-hydroxymuconate, the R39A and R39Q mutations decreased k(cat) by 125- and 389-fold and increased K(m) by 1.5- and 2.6-fold, respectively, suggesting a largely catalytic role for Arg-39. The activity of the R11A/R39A double mutant was at least 10(4)-fold lower than that of the wild-type enzyme, indicating approximate additivity of the effects of the two arginine mutants on k(cat). For both R11A and R39Q, 2D (1)H-(15)N HSQC and 3D (1)H-(15)N NOESY-HSQC spectra showed chemical shift changes mainly near the mutated residues, indicating otherwise intact protein structures. The changes in the R39Q mutant were mainly in the beta-hairpin from residues 50 to 57 which covers the active site. HSQC titration of R11A with the substrate analogue cis, cis-muconate yielded a K(d) of 22 mM, 37-fold greater than the K(d) found with wild-type 4-OT (0.6 mM). With the R39Q mutant, cis, cis-muconate showed negative cooperativity in active site binding with two K(d) values, 3.5 and 29 mM. This observation together with the low K(m) of 2-hydroxymuconate (0.47 mM) suggests that only the tight binding sites function catalytically in the R39Q mutant. The (15)Nepsilon resonances of all six Arg residues of 4-OT were assigned, and the assignments of Arg-11, -39, and -61 were confirmed by mutagenesis. The binding of cis,cis-muconate to wild-type 4-OT upshifts Arg-11 Nepsilon (by 0.05 ppm) and downshifts Arg-39 Nepsilon (by 1.19 ppm), indicating differing electronic delocalizations in the guanidinium groups. A mechanism is proposed in which Arg-11 interacts with the 6-carboxylate of the substrate to facilitate both substrate binding and catalysis and Arg-39 interacts with the 1-carboxylate and the 2-keto group of the substrate to promote carbonyl polarization and catalysis, while Pro-1 transfers protons from C-3 to C-5. This mechanism, together with the effects of mutations of catalytic residues on k(cat), provides a quantitative explanation of the 10(7)-fold catalytic power of 4-OT. Despite its presence in the active site in the crystal structure of the affinity-labeled enzyme, Arg-61 does not play a significant role in either substrate binding or catalysis.     

Andersen's syndrome mutation effects on the structure and assembly of the cytoplasmic domains of Kir2.1

Kir2.1 channels play a key role in maintaining the correct resting potential in eukaryotic cells. Recently, specific amino acid mutations in the Kir2.1 inwardly rectifying potassium channel have been found to cause Andersen's Syndrome in humans. Here, we have characterized individual Andersen's Syndrome mutants R218Q, G300V, E303K, and delta314-315 and have found multiple effects on the ability of the cytoplasmic domains in Kir2.1 channels to form proper tetrameric assemblies. For the R218Q mutation, we identified a second site mutation (T309K) that restored tetrameric assembly but not function. We successfully crystallized and solved the structure (at 2.0 A) of the N- and C-terminal cytoplasmic domains of Kir2.1-R218Q/T309K(S). This new structure revealed multiple conformations of the G-loop and CD loop, providing an explanation for channels that assemble but do not conduct ions. Interestingly, Glu303 forms both intra- and intersubunit salt bridges, depending on the conformation of the G-loop, suggesting that the E303K mutant stabilizes both closed and open G-loop conformations. In the Kir2.1-R218Q/T309K(S) structure, we discovered that the DE loop forms a hydrophobic pocket that binds 2-methyl-2,4-pentanediol, which is located near the putative G(betagamma)-activation site of Kir3 channels. Finally, we observed a potassium ion bound to the cytoplasmic domain for this class of K+ channels.     

Water chain formation and possible proton pumping routes in Rhodobacter sphaeroides cytochrome c oxidase: a molecular dynamics comparison of the wild type and R481K mutant

Cytochrome c oxidase (CcO) converts the energy from redox and oxygen chemistry to support proton translocation and create a transmembrane DeltamuH(+) used for ATP production. Molecular dynamics (MD) simulations were carried out to probe for the formation water chains capable of participating in proton translocation. Attention was focused on the region between and above the a and a(3) hemes where well-defined water chains have not been identified in crystallographic studies. An arginine (R481) (Rhodobacter sphaeroides numbering), positioned between the D-propionates of the hemes, had been mutated in vivo to lysine and showed to have altered activity consistent with an altered proton conductance [Qian, J., Mills, D. A., Geren, L., Wang, K. F., Hoganson, C. W., Schmidt, B., Hiser, C., Babcock, G. T., Durham, B., Millett, F., and Ferguson-Miller, S. (2004) Role of the conserved arginine pair in proton and electron transfer in cytochrome c oxidase, Biochemistry 43, 5748-5756; also see the accompanying paper by Mills et al.]. This mutant was created in silico, and the MD results for the mutant and wild type were compared to explore the effects on the formation of hydrogen-bonded water chains by this mutation. The simulations reveal the presence of hydrogen-bonded water chains that lead from E286 through the region above the hemes to the Mg(2+), and from E286 to the heme a(3) D-propionate and the binuclear center. The R481K mutant does not form as many, or as extensive, water chains as wild-type CcO, due to a new conformation of residues in a large loop between helices III and IV in subunit I, indicating a reduction in the level of water chain formation in the mutant. This loop appears to play a role in controlling the formation of hydrogen-bonded water chains above the hemes. The results suggest a possible gating mechanism for proton movement that includes key residues W172 and Y175 on the loop and F282 on helix VI.     

Connection of transport and sensing by UhpC, the sensor for external glucose-6-phosphate in Escherichia coli.

UhpC is a membrane-bound sensor protein in Escherichia coli required for recognizing external glucose-6-phosphate (Glc6P) and induction of the transport protein UhpT. Recently, it was shown that UhpC is also able to transport Glc6P. In this study we investigated whether these transport and sensing activities are obligatorily coupled in UhpC. We expressed a His-UhpC protein in a UhpC-deficient E. coli strain and verified that this construct does not alter the basic biochemical properties of the Glc6P sensor system. The effects of arginine replacements, mutations of the central loop, and introduction of a salt bridge in UhpC on transport and sensing were compared. The exchanges R46C, R266C and R149C moderately affected transport by UhpC but strongly decreased the sensing ability. This suggested that the affinity for Glc6P as a transported substrate is uncoupled in UhpC from its affinity for Glc6P as an inducer. Four of the 11 arginine mutants showed a constitutive phenotype but had near wild-type transport activity suggesting that Glc6P can be transported by a molecule locked in the inducing conformation. Introduction of an intrahelical salt bridge increased the transport activity of UhpC but abolished sensing. Three conserved residues from the central loop were mutated and although none of these showed transport, one exhibited increased affinity for sensing. Taken together, these data show that transport by UhpC is not required for sensing, that conserved arginine residues are important for sensing and not for transport, and that residues located in the central hydrophilic loop are critical for transport and for sensing.     

R248Q mutation—Beyond p53‐DNA binding

R248 in the DNA binding domain (DBD) of p53 interacts directly with the minor groove of DNA. Earlier nuclear magnetic resonance (NMR) studies indicated that the R248Q mutation resulted in conformation changes in parts of DBD far from the mutation site. However, how information propagates from the mutation site to the rest of the DBD is still not well understood. We performed a series of all‐atom molecular dynamics (MD) simulations to dissect sterics and charge effects of R248 on p53‐DBD conformation: (i) wild‐type p53 DBD; (ii) p53 DBD with an electrically neutral arginine side‐chain; (iii) p53 DBD with R248A; (iv) p53 DBD with R248W; and (v) p53 DBD with R248Q. Our results agree well with experimental observations of global conformational changes induced by the R248Q mutation. Our simulations suggest that both charge‐ and sterics are important in the dynamics of the loop (L3) where the mutation resides. We show that helix 2 (H2) dynamics is altered as a result of a change in the hydrogen bonding partner of D281. In turn, neighboring L1 dynamics is altered: in mutants, L1 predominantly adopts the recessed conformation and is unable to interact with the major groove of DNA. We focused our attention the R248Q mutant that is commonly found in a wide range of cancer and observed changes at the zinc‐binding pocket that might account for the dominant negative effects of R248Q. Furthermore, in our simulations, the S6/S7 turn was more frequently solvent exposed in R248Q, suggesting that there is a greater tendency of R248Q to partially unfold and possibly lead to an increased aggregation propensity. Finally, based on the observations made in our simulations, we propose strategies for the rescue of R248Q mutants. Proteins 2015; 83:2240–2250. © 2015 Wiley Periodicals, Inc.     

Catalytic mechanism of Escherichia coli glycinamide ribonucleotide transformylase probed by site-directed mutagenesis and pH-dependent studies.

Site-directed mutagenesis followed by studies of the pH dependence of the kinetic parameters of the mutants has been used to probe the role of the active site residues and loops in catalysis by glycinamide ribonucleotide transformylase (EC 2.1.2.2). The analysis of the mutants of the strictly conserved active site residues, His108 and Asp144, revealed that His108 acts in a salt bridge with Asp144 as a general acid catalyst with a pK(a) value of 9.7. Asp144 also plays a key role in the preparation of the active site geometry for catalysis. The rate-limiting step in the pH range of 6-10 appears to be the catalytic steps involving tetrahedral intermediates, supported by the observation of a pL (L being H or D)-independent solvent deuterium isotope effect of 2. The ionization of the amino group of glycinamide ribonucleotide both as a free and as a bound form dominates the kinetic behavior at low pH. The analysis of a mutation, H121Q, within the loop spanning amino acids 111-131 suggests the closure of the loop is involved in the binding of the substrate. The kinetic behavior parallels pH effects revealed by a series of X-ray crystallographic structures of the apoenzyme and inhibitor-bound enzyme [Su, Y., Yamashita, M. M., Greasley, S. E. , Mullen, C. A., Shim, J. H., Jennings, P. A., Benkovic, S. J., and Wilson, I. A. (1998) J. Mol. Biol. 281, 485-499], permitting a more exact formulation of the probable catalytic mechanism.     

Amino acid substitutions in the pore helix of GluR6 control inhibition by membrane fatty acids

RNA editing at the Q/R site in the GluR5 and GluR6 subunits of neuronal kainate receptors regulates channel inhibition by lipid-derived modulators including the cis-unsaturated fatty acids arachidonic acid and docosahexaenoic acid. Kainate receptor channels in which all of the subunits are in the edited (R) form exhibit strong inhibition by these compounds, whereas wild-type receptors that include a glutamine (Q) at the Q/R site in one or more subunits are resistant to inhibition. In the present study, we have performed an arginine scan of residues in the pore loop of the GluR6(Q) subunit. Amino acids within the range from -19 to +7 of the Q/R site of GluR6(Q) were individually mutated to arginine and the mutant cDNAs were expressed as homomeric channels in HEK 293 cells. All but one of the single arginine substitution mutants yielded functional channels. Only weak inhibition, typical of wild-type GluR6(Q) channels, was observed for substitutions +1 to +6 downstream of the Q/R site. However, arginine substitution at several locations upstream of the Q/R site resulted in homomeric channels exhibiting strong inhibition by fatty acids, which is characteristic of homomeric GluR6(R) channels. Based on homology with the pore loop of potassium channels, locations at which R substitution induces susceptibility to fatty acid inhibition face away from the cytoplasm toward the M1 and M3 helices and surrounding lipids.     

X-ray, NMR, and mutational studies of the catalytic cycle of the GDP-mannose mannosyl hydrolase reaction

GDP-mannose hydrolase catalyzes the hydrolysis with inversion of GDP-alpha-D-hexose to GDP and beta-D-hexose by nucleophilic substitution by water at C1 of the sugar. Two new crystal structures (free enzyme and enzyme-substrate complex), NMR, and site-directed mutagenesis data, combined with the structure of the enzyme-product complex reported earlier, suggest a four-stage catalytic cycle. An important loop (L6, residues 119-125) contains a ligand to the essential Mg2+ (Gln-123), the catalytic base (His-124), and three anionic residues. This loop is not ordered in the X-ray structure of the free enzyme due to dynamic disorder, as indicated by the two-dimensional 1H-15N HMQC spectrum, which shows selective exchange broadening of the imidazole nitrogen resonances of His-124 (k(ex) = 6.6 x 10(4) s(-1)). The structure of the enzyme-Mg2+-GDP-mannose substrate complex of the less active Y103F mutant shows loop L6 in an open conformation, while the structure of the enzyme-Mg2+-GDP product complex showed loop L6 in a closed, "active" conformation. 1H-15N HMQC spectra show the imidazole N epsilon of His-124 to be unprotonated, appropriate for general base catalysis. Substituting Mg2+ with the more electrophilic metal ions Mn2+ or Co2+ decreases the pKa in the pH versus kcat rate profiles, showing that deprotonation of a metal-bound water is partially rate-limiting. The H124Q mutation, which decreases kcat 10(3.4)-fold and largely abolishes its pH dependence, is rescued by the Y103F mutation, which increases kcat 23-fold and restores its pH dependence. The structural basis of the rescue is the fact that the Y103F mutation shifts the conformational equilibrium to the open form moving loop L6 out of the active site, thus permitting direct access of the specific base hydroxide from the solvent. In the proposed dissociative transition state, which occurs in the closed, active conformation of the enzyme, the partial negative charge of the GDP leaving group is compensated by the Mg2+, and by the closing of loop L2 that brings Arg-37 closer to the beta-phosphate. The development of a positive charge at mannosyl C1, as the oxocarbenium-like transition state is approached, is compensated by closing the anionic loop, L6, onto the active site, further stabilizing the transition state.