Allosteric Inhibition of Human Porphobilinogen Synthase

Porphobilinogen synthase (PBGS) catalyzes the first common step in tetrapyrrole (e.g. heme, chlorophyll) biosynthesis. Human PBGS exists as an equilibrium of high activity octamers, low activity hexamers, and alternate dimer configurations that dictate the stoichiometry and architecture of further assembly. It is posited that small molecules can be found that inhibit human PBGS activity by stabilizing the hexamer. Such molecules, if present in the environment, could potentiate disease states associated with reduced PBGS activity, such as lead poisoning and ALAD porphyria, the latter of which is associated with human PBGS variants whose quaternary structure equilibrium is shifted toward the hexamer (Jaffe, E. K., and Stith, L. (2007) Am. J. Hum. Genet. 80, 329–337). Hexamer-stabilizing inhibitors of human PBGS were identified using in silico prescreening (docking) of ∼111,000 structures to a hexamer-specific surface cavity of a human PBGS crystal structure. Seventy-seven compounds were evaluated in vitro; three provided 90–100% conversion of octamer to hexamer in a native PAGE mobility shift assay. Based on chemical purity, two (ML-3A9 and ML-3H2) were subjected to further evaluation of their effect on the quaternary structure equilibrium and enzymatic activity. Naturally occurring ALAD porphyria-associated human PBGS variants are shown to have an increased susceptibility to inhibition by both ML-3A9 and ML-3H2. ML-3H2 is a structural analog of amebicidal drugs, which have porphyria-like side effects. Data support the hypothesis that human PBGS hexamer stabilization may explain these side effects. The current work identifies allosteric ligands of human PBGS and, thus, identifies human PBGS as a medically relevant allosteric enzyme.     

Single amino acid mutations alter the distribution of human porphobilinogen synthase quaternary structure isoforms (morpheeins)

Porphobilinogen synthase (PBGS) is an obligate oligomer that can exist in functionally distinct quaternary states of different stoichiometries, which are called morpheeins. The morpheein concept describes an ensemble of quaternary structure isoforms wherein different structures of the monomer dictate different multiplicities of the oligomer (Jaffe, E. K. (2005) Trends Biochem. Sci. 30, 490-497). Human PBGS assembles into long-lived morpheeins and has been shown to be capable of forming either a high activity octamer or a low activity hexamer (Breinig, S., Kervinen, J., Stith, L., Wasson, A. S., Fairman, R., Wlodawer, A., Zdanov, A., and Jaffe, E. K. (2003) Nat. Struct. Biol. 10, 757-763). All PBGS monomers contain an alphabeta-barrel domain and an N-terminal arm domain. The N-terminal arm structure varies among PBGS morpheeins, and the spatial relationship between the arm and the barrel dictates the different quaternary assemblies. We have analyzed the structures of human PBGS morpheeins for key interactions that would be predicted to affect the oligomeric assembly. Examples of individual mutations that shift assembly of human PBGS away from the native octamer are R240A and W19A. The alternate morpheeins of human PBGS variants R240A and W19A are chromatographically separable from each other and kinetically distinct; their structure and dynamics have been characterized by native gel electrophoresis, dynamic light scattering, and analytical ultracentrifugation. R240A assembles into a metastable hexamer, which can undergo a reversible conversion to the octamer in the presence of substrate. The metastable nature of the R240A hexamer supports the hypothesis that octameric and hexameric morpheeins of PBGS are very close in energy. W19A assembles into a mixture of dimers, which appear to be stable.     

Kinetics and thermodynamics of the interchange of the morpheein forms of human porphobilinogen synthase

A morpheein is a homo-oligomeric protein that can adopt different nonadditive quaternary assemblies (morpheein forms) with different functionalities. The human porphobilinogen synthase (PBGS) morpheein forms are a high activity octamer, a low activity hexamer, and two structurally distinct dimer conformations. Conversion between hexamer and octamer involves dissociation to dimers, conformational change at the dimer level, followed by association to the alternate assembly. The current work promotes an alternative and novel view of the physiologically relevant dimeric structures, which are derived from the crystal structures, but are distinct from the asymmetric units of their crystal forms. Using a well characterized heteromeric system (WT+F12L; Tang, L. et al. (2005) J. Biol. Chem. 280, 15786-15793), extensive study of the human PBGS morpheein reequilibration process now reveals that the intervening dimers do not dissociate to monomers. The morpheein equilibria of wild type (WT) human PBGS are found to respond to changes in pH, PBGS concentration, and substrate turnover. Notably, the WT enzyme is predominantly an octamer at neutral pH, but increasing pH results in substantial conversion to lower order oligomers. Most significantly, the free energy of activation for the conversion of WT+F12L human PBGS heterohexamers to hetero-octamers is determined to be the same as that for the catalytic conversion of substrate to product by the octamer, remarkably suggesting a common rate-limiting step for both processes, which is postulated to be the opening/closing of the active site lid.     

Probing the oligomeric assemblies of pea porphobilinogen synthase by analytical ultracentrifugation

The enzyme porphobilinogen synthase (PBGS) can exist in different nonadditive homooligomeric assemblies, and under appropriate conditions, the distribution of these assemblies can respond to ligands such as metals or substrate. PBGS from most organisms was believed to be octameric until work on a rare allele of human PBGS revealed an alternate hexameric assembly, which is also available to the wild-type enzyme at elevated pH [Breinig, S., et al. (2003) Nat. Struct. Biol. 10, 757-763]. Herein, we establish that the distribution of pea PBGS quaternary structures also contains octamers and hexamers, using both sedimentation velocity and sedimentation equilibrium experiments. We report results in which the octamer dominates under purification conditions and discuss conditions that influence the octamer:hexamer ratio. As predicted by PBGS crystal structures from related organisms, in the absence of magnesium, the octameric assembly is significantly destabilized, and the oligomeric distribution is dominated largely by the hexameric assembly. Although the PBGS hexamer-to-octamer oligomeric rearrangement is well documented under some conditions, both assemblies are very stable (under AU conditions) in the time frame of our ultracentrifuge experiments.     

Message amplification phenotyping of an inherited delta-aminolevulinate dehydratase deficiency in a family with acute hepatic porphyria

The molecular basis of the enzymatic defect responsible for acute hepatic porphyria due to delta-aminolevulinate dehydratase (ALAD) deficiency was investigated in a family including a proband with the acute disease. In order to delineate the mutation in the proband, cDNA for deficient ALAD was synthesized from the proband's cells. The ALAD phenotype was studied by message amplification phenotyping with total RNA extracted from lymphoblastoid cells of the proband and his family members. Two independent mutant alleles of ALAD were identified in the proband's cells. One mutant allele was shown to result in an amino acid substitution at residue 274 (Ala274----Thr). Message amplification phenotyping studies have also permitted us to define the ALAD phenotype of each subject in the family. This is the first mutation to be recognized in the human ALAD gene.     

An artificial gene for human porphobilinogen synthase allows comparison of an allelic variation implicated in susceptibility to lead poisoning

Porphobilinogen synthase (PBGS) is an ancient enzyme essential to tetrapyrrole biosynthesis (e.g. heme, chlorophyll, and vitamin B(12)). Two common alleles encoding human PBGS, K59 and N59, have been correlated with differential susceptibility of humans to lead poisoning. However, a model for human PBGS based on homologous crystal structures shows the location of the allelic variation to be distant from the active site with its two Zn(II). Previous microbial expression systems for human PBGS have resulted in a poor yield. Here, an artificial gene encoding human PBGS was constructed by recursive polymerase chain reaction from synthetic oligonucleotides to rectify this problem. The artificial gene was made to resemble the highly expressed homologous Escherichia coli hemB gene and to remove rare codons that can confound heterologous protein expression in E. coli. We have expressed and purified recombinant human PBGS variants K59 and N59 in 100-mg quantities. Both human PBGS proteins purified with eight Zn(II)/octamer; Zn(II) binding was shown to be pH-dependent; and Pb(II) could displace some of the Zn(II). However, there was no differential displacement of Zn(II) by Pb(II) between K59 and N59, and simple Pb(II) inhibition studies revealed no allelic difference.     

Role of permissive neuraminidase mutations in influenza A/Brisbane/59/2007-like (H1N1) viruses

Neuraminidase (NA) mutations conferring resistance to NA inhibitors were believed to compromise influenza virus fitness. Unexpectedly, an oseltamivir-resistant A/Brisbane/59/2007 (Bris07)-like H1N1 H275Y NA variant emerged in 2007 and completely replaced the wild-type (WT) strain in 2008-2009. The NA of such variant contained additional NA changes (R222Q, V234M and D344N) that potentially counteracted the detrimental effect of the H275Y mutation on viral fitness. Here, we rescued a recombinant Bris07-like WT virus and 4 NA mutants/revertants (H275Y, H275Y/Q222R, H275Y/M234V and H275Y/N344D) and characterized them in vitro and in ferrets. A fluorometric-based NA assay was used to determine Vmax and Km values. Replicative capacities were evaluated by yield assays in ST6Gal1-MDCK cells. Recombinant NA proteins were expressed in 293T cells and surface NA activity was determined. Infectivity and contact transmission experiments were evaluated for the WT, H275Y and H275Y/Q222R recombinants in ferrets. The H275Y mutation did not significantly alter Km and Vmax values compared to WT. The H275Y/N344D mutant had a reduced affinity (Km of 50 vs 12 μM) whereas the H275Y/M234V mutant had a reduced activity (22 vs 28 U/sec). In contrast, the H275Y/Q222R mutant showed a significant decrease of both affinity (40 μM) and activity (7 U/sec). The WT, H275Y, H275Y/M234V and H275Y/N344D recombinants had comparable replicative capacities contrasting with H275Y/Q222R mutant whose viral titers were significantly reduced. All studied mutations reduced the cell surface NA activity compared to WT with the maximum reduction being obtained for the H275Y/Q222R mutant. Comparable infectivity and transmissibility were seen between the WT and the H275Y mutant in ferrets whereas the H275Y/Q222R mutant was associated with significantly lower lung viral titers. In conclusion, the Q222R reversion mutation compromised Bris07-like H1N1 virus in vitro and in vivo. Thus, the R222Q NA mutation present in the WT virus may have facilitated the emergence of NAI-resistant Bris07 variants.     

Role of arginine 59 in the gamma-class carbonic anhydrases.

The functional role of the highly conserved active site Arg 59 in the prototype of the gamma-class carbonic anhydrase Cam (carbonic anhydrase from Methanosarcina thermophila) was investigated. Variants (R59A, -C, -E, -H, -K, -M, and -Q) were prepared by site-directed mutagenesis and characterized by size exclusion chromatography (SEC), circular dichroism (CD) spectroscopy, and stopped-flow kinetic analyses. CD spectra indicated similar secondary structures for the wild type and the R59A and -K variants, independent of nondenaturing concentrations of guanidine hydrochloride (GdnHCl). SEC indicated that all variants purified as homotrimers like the wild type. SEC also revealed that the R59A and -K variants unfolded at > or = 1.5 M GdnHCl, compared to 3.0 M GdnHCl for the wild type. These results indicate that Arg 59 contributes to the thermodynamic stability of the Cam trimer. The R59K variant had k(cat) and k(cat)/K(m) values that were 8 and 5% of the wild-type values, respectively, while all other variants had k(cat) and k(cat)/K(m) values 10-100-fold lower than those of the wild type. The R59A, -C, -E, -M, and -Q variants exhibited 4-63-fold increases in k(cat) and 9-120-fold increases in k(cat)/K(m) upon addition of 100 mM GdnHCl, with the largest increases observed for the R59A variant, which was comparable to the R59K variant. The kinetic results indicate that a positive charge at position 59 is essential for the CO(2) hydration step of the overall catalytic mechanism.     

Functional characterization of genetic variants of human FMO3 associated with trimethylaminuria

Impaired conversion of trimethylamine to trimethylamine N-oxide by human flavin containing monooxygenase 3 (FMO3) is strongly associated with primary trimethylaminuria, also known as 'fish-odor' syndrome. Numerous non-synonymous mutations in FMO3 have been identified in patients suffering from this metabolic disorder (e.g., N61S, M66I, P153L, and R492W), but the molecular mechanism(s) underlying the functional deficit attributed to these alleles has not been elucidated. The purpose of the present study was to determine the impact of these disease-associated genetic variants on FMO3 holoenzyme formation and on steady-state kinetic parameters for metabolism of several substrates, including trimethylamine. For comparative purposes, several common allelic variants not associated with primary trimethylaminuria (i.e., E158K, V257M, E308G, and the E158K/E308G haplotype) were also analyzed. When recombinantly expressed in insect cells, only the M66I and R492W mutants failed to incorporate/retain the FAD cofactor. Of the remaining mutant proteins P153L and N61S displayed substantially reduced (<10%) catalytic efficiencies for trimethylamine N-oxygenation relative to the wild-type enzyme. For N61S, reduced catalytic efficiency was solely a consequence of an increased K(m), whereas for P153L, both K(m) and k(cat) were altered. Similar results were obtained when benzydamine N-oxygenation was monitored. A homology model for FMO3 was constructed based on the crystal structure for yeast FMO which places the N61 residue alone, of the mutants analyzed here, in close proximity to the FAD catalytic center. These data demonstrate that primary trimethylaminuria is multifactorial in origin in that enzyme dysfunction can result from kinetic incompetencies as well as impaired assembly of holoprotein.     

Molecular analysis in Brazilian cystic fibrosis patients reveals five novel mutations

We have performed molecular genetic analyses on 160 Brazilian patients diagnosed with cystic fibrosis (CF). Screening of mutations in 320 CF chromosomes was performed through single strand conformation polymorphism (SSCP) and heteroduplex analyses assay followed by DNA sequencing of the 27 exons and exon/intron boundaries of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The frequency of CFTR variants of T-tract length of intron 8 (IVS8 Tn) was also investigated. This analysis enabled the detection of 232/320 CF mutations (72.2%) and complete genotyping of 61% of the patients. The deltaF508 mutation was found in 48.4% of the alleles. Another fifteen mutations (previously reported) were detected: G542X, R1162X, N1303K, R334W, W1282X, G58E, L206W, R553X, 621+1G-->T, V232D, 1717-1G-->A, 2347 delG, R851L, 2789+5G-->A, and W1089X. Five novel mutations were identified, V201M (exon 6a), Y275X (exon 6b), 2686 insT (exon 14a), 3171 delC (exon 17a), and 3617 delGA (exon 19). These results contribute to the molecular characterization of CF in the Brazilian population. In addition, the identification of the novel mutation Y275X allowed prenatal diagnosis in a high-risk fetus.     

Redox and metal-regulated oligomeric state for human porphobilinogen synthase activation

The oligomeric state of human porphobilinogen synthase (PBGS) [EC.4.2.1.24] is homooctamer, which consists of conformationally heterogenous subunits in the tertiary structure under air-saturated conditions. When PBGS is activated by reducing agent with zinc ion, a reservoir zinc ion coordinated by Cys²²³ is transferred in the active center to be coordinated by Cys¹²², Cys¹²⁴, and Cys¹³² (Sawada et al. in J Biol Inorg Chem 10:199–207, 2005). The latter zinc ion serves as an electrophilic catalysis. In this study, we investigated a conformational change associated with the PBGS activation by reducing agent and zinc ion using analytical ultracentrifugation, negative staining electron microscopy, native PAGE, and enzyme activity staining. The results are in good agreement with our notion that the main component of PBGS is octamer with a few percent of hexamer and that the octamer changes spatial subunit arrangement upon reduction and further addition of zinc ion, accompanying decrease in f/f ₀. It is concluded that redox-regulated PBGS activation via cleavage of disulfide bonds among Cys¹²², Cys¹²⁴, and Cys¹³² and coordination with zinc ion is closely linked to change in the oligomeric state.     

Impact of Potential Permissive Neuraminidase Mutations on Viral Fitness of the H275Y Oseltamivir-Resistant Influenza A(H1N1)pdm09 Virus In Vitro,in Mice and in Ferrets

Neuraminidase (NA) mutations conferring resistance to NA inhibitors (NAIs) generally compromise the fitness of influenza viruses. The only NAI-resistant virus that widely spread in the population, the A/Brisbane/59/2007 (H1N1) strain, contained permissive mutations that restored the detrimental effect caused by the H275Y change. Computational analysis predicted other permissive NA mutations for A(H1N1)pdm09 viruses. Here, we investigated the effect of T289M and N369K mutations on the viral fitness of the A(H1N1)pdm09 H275Y variant. Recombinant wild-type (WT) A(H1N1)pdm09 and the H275Y, H275Y/T289M, H275Y/N369K, and H275Y/V241I/N369K (a natural variant) NA mutants were generated by reverse genetics. Replication kinetics were performed by using ST6GalI-MDCK cells. Virulence was assessed in C57BL/6 mice, and contact transmission was evaluated in ferrets. The H275Y mutation significantly reduced viral titers during the first 12 to 36 h postinfection (p.i.) in vitro. Nevertheless, the WT and H275Y viruses induced comparable mortality rates, weight loss, and lung titers in mice. The T289M mutation eliminated the detrimental effect caused by the H275Y change in vitro while causing greater weight loss and mortality in mice, with significantly higher lung viral titers on days 3 and 6 p.i. than with the H275Y mutant. In index ferrets, the WT, H275Y, H275Y/T289M, and H275Y/V241I/N369K recombinants induced comparable fever, weight loss, and nasal wash viral titers. All tested viruses were transmitted at comparable rates in contact ferrets, with the H275Y/V241I/N369K recombinant demonstrating higher nasal wash viral titers than the H275Y mutant. Permissive mutations may enhance the fitness of A(H1N1)pdm09 H275Y viruses in vitro and in vivo. The emergence of such variants should be carefully monitored.     

Control of tetrapyrrole biosynthesis by alternate quaternary forms of porphobilinogen synthase

Porphobilinogen synthase (PBGS) catalyzes the first common step in the biosynthesis of tetrapyrroles (such as heme and chlorophyll). Although the predominant oligomeric form of this enzyme, as inferred from many crystal structures, is that of a homo-octamer, a rare human PBGS allele, F12L, reveals the presence of a hexameric form. Rearrangement of an N-terminal arm is responsible for this oligomeric switch, which results in profound changes in kinetic behavior. The structural transition between octamer and hexamer must proceed through an unparalleled equilibrium containing two different dimer structures. The allosteric magnesium, present in most PBGS, has a binding site in the octamer but not in the hexamer. The unprecedented structural rearrangement reported here relates to the allosteric regulation of PBGS and suggests that alternative PBGS oligomers may function in a magnesium-dependent regulation of tetrapyrrole biosynthesis in plants and some bacteria.     

Familial Mediterranean fever in Syrian patients: <Emphasis Type="Italic">MEFV</Emphasis> gene mutations and genotype–phenotype correlation

Familial Mediterranean fever is an autosomal recessive disorder characterized by recurrent attacks of abdominal pain, synovitis and pleuritis. MEFV gene mutations are responsible for the disease. The objective of this study was to identify the frequency and distribution of 12 MEFV mutations in 153 Syrian patients and perform a genotype–phenotype correlation in the patients’ cohort. Of the 153 unrelated patients investigated, 97 (63.4%) had at least one mutation. The most frequent mutation was M694V (36.5%), followed by V726A (15.2%), E148Q (14.5%), M680I (G/C) (13.2%), and M694I (10.2%) mutations. Rare mutations (R761H, A744S, M680I (G/A), K695R, P369S, F479L and I692del) were also detected in the patients. M694V was associated with the severe form of the disease. The identification of a significant number of FMF patients with no mutations or only one known mutation identified indicates the presence of new mutations in the MEFV gene which will be investigated in the future.     

Morpheeins--a new structural paradigm for allosteric regulation

Classic models for the allosteric regulation of protein function consider an equilibrium among protein structures of constant oligomeric multiplicity. The morpheein (mor-phee'-in) concept expands this model to include a dynamic equilibrium of protein structures wherein a protein monomer can exist in more than one conformation and each monomer conformation dictates a different quaternary structure of finite multiplicity and different functionality. The morpheein concept provides a new framework for understanding allosteric regulation, kinetic cooperativity and hysteresis. Porphobilinogen synthase constitutes a prototype morpheein ensemble comprising several interconverting quaternary structure isoforms; one monomer conformation dictates assembly of a high-activity octamer, whereas an alternative monomer conformation dictates assembly of a low-activity hexamer. It is proposed here that the behavior of some other allosteric enzymes reflect dynamic morpheein equilibrium systems and six candidate proteins are enumerated.     

Reaction mechanism of glutathione synthetase from Arabidopsis thaliana: site-directed mutagenesis of active site residues

Glutathione is essential for maintaining the intracellular redox environment and is synthesized from gamma-glutamylcysteine, glycine, and ATP by glutathione synthetase (GS). To examine the reaction mechanism of a eukaryotic GS, 24 Arabidopsis thaliana GS (AtGS) mutants were kinetically characterized. Within the gamma-glutamylcysteine/glutathione-binding site, the S153A and S155A mutants displayed less than 4-fold changes in kinetic parameters with mutations of Glu-220 (E220A/E220Q), Gln-226 (Q226A/Q226N), and Arg-274 (R274A/R274K) at the distal end of the binding site resulting in 24-180-fold increases in the K(m) values for gamma-glutamylcysteine. Substitution of multiple residues interacting with ATP (K313M, K367M, and E429A/E429Q) or coordinating magnesium ions to ATP (E148A/E148Q, N150A/N150D, and E371A) yielded inactive protein because of compromised nucleotide binding, as determined by fluorescence titration. Other mutations in the ATP-binding site (E371Q, N376A, and K456M) resulted in greater than 30-fold decreases in affinity for ATP and up to 80-fold reductions in turnover rate. Mutation of Arg-132 and Arg-454, which are positioned at the interface of the two substrate-binding sites, affected the enzymatic activity differently. The R132A mutant was inactive, and the R132K mutant decreased k(cat) by 200-fold; however, both mutants bound ATP with K(d) values similar to wild-type enzyme. Minimal changes in kinetic parameters were observed with the R454K mutant, but the R454A mutant displayed a 160-fold decrease in k(cat). In addition, the R132K, R454A, and R454K mutations elevated the K(m) value for glycine up to 11-fold. Comparison of the pH profiles and the solvent deuterium isotope effects of A. thaliana GS and the Arg-132 and Arg-454 mutants also suggest distinct mechanistic roles for these residues. Based on these results, a catalytic mechanism for the eukaryotic GS is proposed.     

Conformational stability and activity analysis of two hydroxymethylbilane synthase mutants,K132N and V215E,with different phenotypic association with acute intermittent porphyria

The autosomal dominantly inherited disease AIP (acute intermittent porphyria) is caused by mutations in HMBS [hydroxymethylbilane synthase; also known as PBG (porphobilinogen) deaminase], the third enzyme in the haem biosynthesis pathway. Enzyme-intermediates with increasing number of PBG molecules are formed during the catalysis of HMBS. In this work, we studied the two uncharacterized mutants K132N and V215E comparative with wt (wild-type) HMBS and to the previously reported AIP-associated mutants R116W, R167W and R173W. These mainly present defects in conformational stability (R116W), enzyme kinetics (R167W) or both (R173W). A combination of native PAGE, CD, DSF (differential scanning fluorimetry) and ion-exchange chromatography was used to study conformational stability and activity of the recombinant enzymes. We also investigated the distribution of intermediates corresponding to specific elongation stages. It is well known that the thermostability of HMBS increases when the DPM (dipyrromethane) cofactor binds to the apoenzyme and the holoenzyme is formed. Interestingly, a decrease in thermal stability was measured concomitant to elongation of the pyrrole chain, indicating a loosening of the structure prior to product release. No conformational or kinetic defect was observed for the K132N mutant, whereas V215E presented lower conformational stability and probably a perturbed elongation process. This is in accordance with the high association of V215E with AIP. Our results contribute to interpret the molecular mechanisms for dysfunction of HMBS mutants and to establish genotype–phenotype relations for AIP.     

Structure-function analysis of Friedreich's ataxia mutants reveals determinants of frataxin binding and activation of the Fe-S assembly complex

Friedreich's ataxia (FRDA) is a progressive neurodegenerative disease associated with the loss of function of the protein frataxin (FXN) that results from low FXN levels due to a GAA triplet repeat expansion or, occasionally, from missense mutations in the FXN gene. Here biochemical and structural properties of FXN variants, including three FRDA missense mutations (N146K, Q148R, and R165C) and three related mutants (N146A, Q148G, and Q153A), were determined in an effort to understand the structural basis for the loss of function. In vitro assays revealed that although the three FRDA missense mutations exhibited similar losses of cysteine desulfurase and Fe-S cluster assembly activities, the causes for these activation defects were distinct. The R165C variant exhibited a k(cat)/K(M) higher than that of native FXN but weak binding to the NFS1, ISD11, and ISCU2 (SDU) complex, whereas the Q148R variant exhibited the lowest k(cat)/K(M) of the six tested FXN variants and only a modest binding deficiency. The order of the FXN binding affinities for the SDU Fe-S assembly complex was as follows: FXN > Q148R > N146A > Q148G > N146K > Q153A > R165C. Four different classes of FXN variants were identified on the basis of their biochemical properties. Together, these structure-function studies reveal determinants for the binding and allosteric activation of the Fe-S assembly complex and provide insight into how FRDA missense mutations are functionally compromised.     

GABA(A) receptor M2-M3 loop secondary structure and changes in accessibility during channel gating

The gamma-aminobutyric acid type A (GABA(A)) receptor M2-M3 loop structure and its role in gating were investigated using the substituted cysteine accessibility method. Residues from alpha(1)Arg-273 to alpha(1)Ile-289 were mutated to cysteine, one at a time. MTSET(+) or MTSES(-) reacted with all mutants from alpha(1)R273C to alpha(1)Y281C, except alpha(1)P277C, in the absence and presence of GABA. The MTSET(+) closed-state reaction rate was >1000 liters/mol-s at alpha(1)N274C, alpha(1)S275C, alpha(1)K278C, and alpha(1)Y281C and was <300 liters/mol-s at alpha(1)R273C, alpha(1)L276C, alpha(1)V279C, alpha(1)A280C, and alpha(1)A284C. These two groups of residues lie on opposite sides of an alpha-helix. The fast reacting group lies on a continuation of the M2 segment channel-lining helix face. This suggests that the M2 segment alpha-helix extends about two helical turns beyond alpha(1)N274 (20'), aligned with the extracellular ring of charge. At alpha(1)S275C, alpha(1)V279C, alpha(1)A280C, and alpha(1)A284C the reaction rate was faster in the presence of GABA. The reagents had no functional effect on the mutants from alpha(1)A282C to alpha(1)I289C, except alpha(1)A284C. Access may be sterically hindered possibly by close interaction with the extracellular domain. We suggest that the M2 segment alpha-helix extends beyond the predicted extracellular end of the M2 segment and that gating induces a conformational change in and/or around the N-terminal half of the M2-M3 loop. Implications for coupling ligand-evoked conformational changes in the extracellular domain to channel gating in the membrane-spanning domain are discussed.     

Mechanistic implications of mutations to the active site lysine of porphobilinogen synthase

Porphobilinogen synthase (PBGS) is a homo-octameric protein that catalyzes the complex asymmetric condensation of two molecules of 5-aminolevulinic acid (ALA). The only characterized intermediate in the PBGS-catalyzed reaction is a Schiff base that forms between the first ALA that binds and a conserved lysine, which in Escherichia coli PBGS is Lys-246 and in human PBGS is Lys-252. In this study, E. coli PBGS mutants K246H, K246M, K246W, K246N, and K246G and human PBGS mutant K252G were characterized. Alterations to this lysine result in a disabled but not totally inactive protein suggesting an alternate mechanism in which proximity and orientation are major catalytic devices. (13)C NMR studies of [3,5-(13)C]porphobilinogen bound at the active sites of the E. coli PBGS and the mutants show only minor chemical shift differences, i.e. environmental alterations. Mammalian PBGS is established to have four functional active sites, whereas the crystal structure of E. coli PBGS shows eight spatially distinct and structurally equivalent subunits. Biochemical data for E. coli PBGS have been interpreted to support both four and eight active sites. A unifying hypothesis is that formation of the Schiff base between this lysine and ALA triggers a conformational change that results in asymmetry. Product binding studies with wild-type E. coli PBGS and K246G demonstrate that both bind porphobilinogen at four per octamer although the latter cannot form the Schiff base from substrate. Thus, formation of the lysine to ALA Schiff base is not required to initiate the asymmetry that results in half-site reactivity.