Ligation events influence ALG-2 dimerization.

ALG-2 dimerization was studied using Förster resonance-energy-transfer. D162C variants of ALG-2des23 were covalently modified with Alexa Fluor 488 and Alexa Fluor 647. When samples of the two labeled protein-preparations are combined, the sensitized emission from AF647 serves as a sensitive probe of dimer formation. At 25 °C, in the absence of divalent ions, the wild-type homodimer, ΔGF122 homodimer, and heterodimer display dissociation constants of 7.1, 26, and 4.5 nM, respectively. At 35 °C, subunit interaction is weaker, indicating that dimer formation is exothermic. Binding of Mg2+ in the C-terminal EF-hand (EF5) dramatically enhances ALG-2 dimer stability. Although occupation of EF5 by Ca2+ likewise has a stabilizing effect, its direct influence on dimer stability would be negligible at cytosolic Ca2+ levels. However, dimer stability is substantially increased by the Ca2+-dependent binding of ALG-2 target-peptides, suggesting that the occupation-status of the target-protein binding site is communicated to the dimer interface. Tween 20 is commonly used to improve ALG-2 solubility, the micelles ostensibly acting as target-protein surrogates. Paradoxically, however, the detergent markedly destabilizes ALG-2 dimers, particularly in the presence of Ca2+.

Structural Investigation of a Dimeric Variant of Pyruvate Kinase Muscle Isoform 2.

Pyruvate kinase muscle isoform 2 (PKM2) catalyzes the terminal step in glycolysis, transferring a phosphoryl group from phosphoenolpyruvate to ADP, to produce pyruvate and ATP. PKM2 activity is allosterically regulated by fructose 1,6-bisphosphate (FBP), an upstream glycolytic intermediate. FBP stabilizes the tetrameric form of the enzyme. In its absence, the PKM2 tetramers dissociate, yielding a dimer-monomer mixture having lower enzymatic activity. The S437Y variant of PKM2 is incapable of binding FBP. Consistent with that defect, we find that S437Y exists in a monomer-dimer equilibrium in solution, with a Kd of ∼20 µM. Interestingly, however, the protein crystallizes as a tetramer, providing insight into the structural basis for impaired FBP binding of S437Y.

Importance of the C-Terminus of Aldehyde Dehydrogenase 7A1 for Oligomerization and Catalytic Activity.

Aldehyde dehydrogenase 7A1 (ALDH7A1) catalyzes the terminal step of lysine catabolism, the NAD+-dependent oxidation of α-aminoadipate semialdehyde to α-aminoadipate. Structures of ALDH7A1 reveal the C-terminus is a gate that opens and closes in response to the binding of α-aminoadipate. In the closed state, the C-terminus of one protomer stabilizes the active site of the neighboring protomer in the dimer-of-dimers tetramer. Specifically, Ala505 and Gln506 interact with the conserved aldehyde anchor loop structure in the closed state. The apparent involvement of these residues in catalysis is significant because they are replaced by Pro505 and Lys506 in a genetic deletion (c.1512delG) that causes pyridoxine-dependent epilepsy. Inspired by the c.1512delG defect, we generated variant proteins harboring either A505P, Q506K, or both mutations (A505P/Q506K). Additionally, a C-terminal truncation mutant lacking the last eight residues was prepared. The catalytic behaviors of the variants were examined in steady-state kinetic assays, and their quaternary structures were examined by analytical ultracentrifugation. The mutant enzymes exhibit a profound kinetic defect characterized by markedly elevated Michaelis constants for α-aminoadipate semialdehyde, suggesting that the mutated residues are important for substrate binding. Furthermore, analyses of the in-solution oligomeric states revealed that the mutant enzymes are defective in tetramer formation. Overall, these results suggest that the C-terminus of ALDH7A1 is crucial for the maintenance of both the oligomeric state and the catalytic activity.

Structure and characterization of a class 3B proline utilization A: Ligand-induced dimerization and importance of the C-terminal domain for catalysis.

The bifunctional flavoenzyme proline utilization A (PutA) catalyzes the two-step oxidation of proline to glutamate using separate proline dehydrogenase (PRODH) and l-glutamate-γ-semialdehyde dehydrogenase active sites. Because PutAs catalyze sequential reactions, they are good systems for studying how metabolic enzymes communicate via substrate channeling. Although mechanistically similar, PutAs vary widely in domain architecture, oligomeric state, and quaternary structure, and these variations represent different structural solutions to the problem of sequestering a reactive metabolite. Here, we studied PutA from Corynebacterium freiburgense (CfPutA), which belongs to the uncharacterized 3B class of PutAs. A 2.7 Šresolution crystal structure showed the canonical arrangement of PRODH, l-glutamate-γ-semialdehyde dehydrogenase, and C-terminal domains, including an extended interdomain tunnel associated with substrate channeling. The structure unexpectedly revealed a novel open conformation of the PRODH active site, which is interpreted to represent the non-activated conformation, an elusive form of PutA that exhibits suboptimal channeling. Nevertheless, CfPutA exhibited normal substrate-channeling activity, indicating that it isomerizes into the active state under assay conditions. Sedimentation-velocity experiments provided insight into the isomerization process, showing that CfPutA dimerizes in the presence of a proline analog and NAD+ These results are consistent with the morpheein model of enzyme hysteresis, in which substrate binding induces conformational changes that promote assembly of a high-activity oligomer. Finally, we used domain deletion analysis to investigate the function of the C-terminal domain. Although this domain contains neither catalytic residues nor substrate sites, its removal impaired both catalytic activities, suggesting that it may be essential for active-site integrity.

Impact of disease-Linked mutations targeting the oligomerization interfaces of aldehyde dehydrogenase 7A1.

Aldehyde dehydrogenase 7A1 (ALDH7A1) is involved in lysine catabolism, catalyzing the oxidation of α-aminoadipate semialdehyde to α-aminoadipate. Certain mutations in the ALDH7A1 gene, which are presumed to reduce catalytic activity, cause an autosomal recessive seizure disorder known as pyridoxine-dependent epilepsy (PDE). Although the genetic association between ALDH7A1 and PDE is well established, little is known about the impact of PDE-mutations on the structure and catalytic function of the enzyme. Herein we report the first study of the molecular consequences of PDE mutations using purified ALDH7A1 variants. Eight variants, with mutations in the oligomer interfaces, were expressed in Escherichia coli: P78L, G83E, A129P, G137V, G138V, A149E, G255D, and G263E. All but P78L and G83E were soluble and could be purified. All six soluble mutants were catalytically inactive. The impact of the mutations on oligomerization was assessed by analytical ultracentrifugation. Wild-type ALDH7A1 is shown to exist in a dimer-tetramer equilibrium with a dissociation constant of 16 µM. In contrast to the wild-type enzyme, the variants reside in monomer-dimer equilibria and are apparently incapable of forming a tetrameric species, even at high enzyme concentration. The available evidence suggests that they are misfolded assemblies lacking the three-dimensional structure required for catalysis.

EF5 Is the High-Affinity Mg(2+) Site in ALG-2.

The penta-EF-hand (PEF) protein ALG-2 (apoptosis-linked gene 2) has been implicated in several important physiological processes, including endoplasmic reticulum-Golgi vesicular transport and endosomal biogenesis/transport. ALG-2 was recently shown to harbor a metal ion-binding site with a high affinity for Mg(2+) and a low affinity for Ca(2+). We herein present the X-ray structure of Mg(2+)-bound ALG-2des23(wt). Although the C(α) trace is nearly indistinguishable from that of the Ca(2+)-free protein, the orientation of the C-terminal helix differs in the two structures. Consistent with that observation, replacement of the +x ligand in EF5, D169, with alanine eliminates high-affinity Mg(2+) binding. It also eliminates the low-affinity Ca(2+) site and lowers the affinity of the remaining Ca(2+)-binding sites, EF3 and EF1. The coordination environment in EF5 approaches ideal Mg(2+) octahedral geometry. The ligand array, consisting of three carboxylates (+x, +y, +z), a backbone carbonyl (-y), and two water molecules (-x, -z), may offer a recipe for a high-affinity, high-selectivity Mg(2+)-binding site. Sequence data for other PEF proteins indicate that select calpain large subunits, notably CAPN1 and CAPN8, may also possess a high-affinity Mg(2+)-binding site. In Mg(2+)-bound ALG-2, the carbonyl of F188 and the C-terminal carboxylate of V191 interact with the ε-ammonium group of K137 in the opposing subunit, suggesting that Mg(2+) binding could have an impact on dimerization. Interestingly, EF1 and EF3 are also occupied in the crystal, despite having modest affinity for Mg(2+). The results of a calorimetry-based analysis indicate that their Mg(2+) binding constants are 2 orders of magnitude lower than that determined for EF5.

Diethylaminobenzaldehyde is a covalent, irreversible inactivator of ALDH7A1.

There is growing interest in aldehyde dehydrogenases (ALDHs) because of their overexpression in cancer stem cells and the ability to mediate resistance to cancer drugs. Here, we report the first crystal structure of an aldehyde dehydrogenase complexed with the inhibitor 4-diethylaminobenzaldehyde (DEAB). Contrary to the widely held belief that DEAB is a reversible inhibitor of ALDHs, we show that DEAB irreversibly inactivates ALDH7A1 via formation of a stable, covalent acyl-enzyme species.

Site-directed mutagenesis of rat α-parvalbumin: replacement of canonical CD-site residues with their non-consensus counterparts from rat ß-parvalbumin.

Rat ß-parvalbumin (ß-PV) displays low divalent-ion affinity. Its CD site is distinguished by six non-consensus residues--the "CD-loop residues"--at positions 49, 50, 57-60. Additionally, leucine occupies position 85, rather than phenylalanine, the ß-lineage-consensus residue. Replacement of the CD-loop residues in rat ß with the canonical residues was previously found to have little effect on divalent-ion affinity, unless L85 is replaced by phenylalanine. Herein, we replace the canonical CD-loop residues in rat α-PV with their rat ß-PV counterparts. Although the mutations have a generally modest impact on affinity, E59D confers Ca(2+)-specificity on the CD site, in the presence or absence of the other mutations. Despite their minimal impact on ΔG, several CD-loop mutations markedly alter ΔH, evidently by perturbing the apo-protein conformation. The L85F mutation was also examined. In wild-type rat α, L85F increases EF-site Ca(2+) affinity. In the CD-loop variants, the mutation leaves the ΔG for Ca(2+)-binding largely unaffected. However, several variants display highly exothermic binding enthalpies, indicative of ligation-linked protein-folding. Consistent with that idea, scanning-calorimetry data confirm that L85F has significantly destabilized those proteins.

Structural studies of yeast Δ(1)-pyrroline-5-carboxylate dehydrogenase (ALDH4A1): active site flexibility and oligomeric state.

The proline catabolic enzyme Δ(1)-pyrroline-5-carboxylate dehydrogenase (ALDH4A1) catalyzes the NAD(+)-dependent oxidation of γ-glutamate semialdehyde to l-glutamate. In Saccharomyces cerevisiae, ALDH4A1 is encoded by the PUT2 gene and known as Put2p. Here we report the steady-state kinetic parameters of the purified recombinant enzyme, two crystal structures of Put2p, and the determination of the oligomeric state and quaternary structure from small-angle X-ray scattering and sedimentation velocity. Using Δ(1)-pyrroline-5-carboxylate as the substrate, catalytic parameters kcat and Km were determined to be 1.5 s(-1) and 104 µM, respectively, with a catalytic efficiency of 14000 M(-1) s(-1). Although Put2p exhibits the expected aldehyde dehydrogenase superfamily fold, a large portion of the active site is disordered in the crystal structure. Electron density for the 23-residue aldehyde substrate-binding loop is absent, implying substantial conformational flexibility in solution. We furthermore report a new crystal form of human ALDH4A1 (42% identical to Put2p) that also shows disorder in this loop. The crystal structures provide evidence of multiple active site conformations in the substrate-free form of the enzyme, which is consistent with a conformational selection mechanism of substrate binding. We also show that Put2p forms a trimer-of-dimers hexamer in solution. This result is unexpected because human ALDH4A1 is dimeric, whereas some bacterial ALDH4A1s are hexameric. Thus, global sequence identity and domain of life are poor predictors of the oligomeric states of ALDH4A1. Mutation of a single Trp residue that forms knob-in-hole interactions across the dimer-dimer interface abrogates hexamer formation, suggesting that this residue is the center of a protein-protein association hot spot.

Characterization of avian thymic hormone and chicken parvalbumin 3 target cells.

Avian thymic hormone (ATH) is a ß-parvalbumin produced by epithelial cells in the thymic cortex and in the eyes of chickens. Chicken parvalbumin 3 (CPV3) is a homologous protein produced in the thymus and in hair cells of the chicken ear. ATH circulates in the blood on a five-day cycle and stimulates cell-mediated immunity when administered to young chickens. We report the identification of target cells for ATH and CPV3 and the immunophenotype of target cells for ATH. Newly hatched chicks were injected intracoelomically with ATH and killed 5, 10, 15 or 20 min later. Naïve chickens also were killed at 1, 7 and 14 days of age. Various tissues were examined by EM for the presence of either ATH or CPV3 using colloidal gold labeling. Gold particles were initially present on plasma membranes of lymphocytes in T cell areas of spleen and cecal tonsils from the chicks injected with ATH, internalized within 10 min, and accumulated in nuclei by 20 min. Immunofluorescence staining also identified the presence of ATH in T cell areas of spleen and cecal tonsils. Target cells labeled for ATH were immunophenotyped by double labeling. They were positive for CD3, CD8 and the lymphocyte receptor TCR-1, a phenotype characteristic of cytotoxic γδ T cells. Some of the target cells in the spleen were TCR-3 positive. Targeting of lymphocytes by CPV3 indicated that it may also be an immunomodulating hormone.

Simultaneous addition of two ligands: a potential strategy for estimating divalent ion affinities in EF-hand proteins by isothermal titration calorimetry.

Capable of providing a detailed thermodynamic picture of noncovalent association reactions, isothermal titration calorimetry (ITC) has become a popular method for studying protein-ligand interactions. We routinely employ the technique to study divalent ion-binding by two-site EF-hand proteins from the parvalbumin- and polcalcin lineages. The combination of high Ca(2+) affinity and relatively low Mg(2+) affinity, and the attendant complication of parameter correlation, conspire to make the simultaneous extraction of binding constants and -enthalpies for both ions challenging. Although global analysis of multiple ITC experiments can overcome these hurdles, our current experimental protocol includes upwards of 10 titrations - requiring a substantial investment in labor, machine time, and material. This paper explores the potential for using a smaller suite of experiments that includes simultaneous titrations with Ca(2+) and Mg(2+) at different ratios of the two ions. The results obtained for four proteins, differing substantially in their divalent ion-binding properties, suggest that the approach has merit. The Ca(2+)- and Mg(2+)-binding constants afforded by the streamlined analysis are in reasonable agreement with those obtained from the standard analysis protocol. Likewise, the abbreviated analysis provides comparable values for the Ca(2+)-binding enthalpies. However, the streamlined analysis can yield divergent values for the Mg(2+)-binding enthalpies - particularly those for lower affinity sites. This shortcoming can be remedied, in large measure, by including data from a direct Ca(2+) titration in the presence of a high, fixed Mg(2+) concentration.

Solution structures of polcalcin Phl p 7 in three ligation states: Apo-, hemi-Mg2+-bound, and fully Ca2+-bound.

Polcalcins are small EF-hand proteins believed to assist in regulating pollen-tube growth. Phl p 7, from timothy grass (Phleum pratense), crystallizes as a domain-swapped dimer at low pH. This study describes the solution structures of the recombinant protein in buffered saline at pH 6.0, containing either 5.0 mM EDTA, 5.0 mM Mg(2+), or 100 µM Ca(2+). Phl p 7 is monomeric in all three ligation states. In the apo-form, both EF-hand motifs reside in the closed conformation, with roughly antiparallel N- and C-terminal helical segments. In 5.0 mM Mg(2+), the divalent ion is bound by EF-hand 2, perturbing interhelical angles and imposing more regular helical structure. The structure of Ca(2+)-bound Phl p 7 resembles that previously reported for Bet v 4-likewise exposing apolar surface to the solvent. Occluded in the apo- and Mg(2+)-bound forms, this surface presumably provides the docking site for Phl p 7 targets. Unlike Bet v 4, EF-hand 2 in Phl p 7 includes five potential anionic ligands, due to replacement of the consensus serine residue at -x (residue 55 in Phl p 7) with aspartate. In the Phl p 7 crystal structure, D55 functions as a helix cap for helix D. In solution, however, D55 apparently serves as a ligand to the bound Ca(2+). When Mg(2+) resides in site 2, the D55 carboxylate withdraws to a distance consistent with a role as an outer-sphere ligand. (15)N relaxation data, collected at 600 MHz, indicate that backbone mobility is limited in all three ligation states.

The three-dimensional structural basis of type II hyperprolinemia.

Type II hyperprolinemia is an autosomal recessive disorder caused by a deficiency in Δ(1)-pyrroline-5-carboxylate dehydrogenase (P5CDH; also known as ALDH4A1), the aldehyde dehydrogenase that catalyzes the oxidation of glutamate semialdehyde to glutamate. Here, we report the first structure of human P5CDH (HsP5CDH) and investigate the impact of the hyperprolinemia-associated mutation of Ser352 to Leu on the structure and catalytic properties of the enzyme. The 2. 5-Å-resolution crystal structure of HsP5CDH was determined using experimental phasing. Structures of the mutant enzymes S352A (2.4 Å) and S352L (2.85 Å) were determined to elucidate the structural consequences of altering Ser352. Structures of the 93% identical mouse P5CDH complexed with sulfate ion (1.3 Å resolution), glutamate (1.5 Å), and NAD(+) (1.5 Å) were determined to obtain high-resolution views of the active site. Together, the structures show that Ser352 occupies a hydrophilic pocket and is connected via water-mediated hydrogen bonds to catalytic Cys348. Mutation of Ser352 to Leu is shown to abolish catalytic activity and eliminate NAD(+) binding. Analysis of the S352A mutant shows that these functional defects are caused by the introduction of the nonpolar Leu352 side chain rather than the removal of the Ser352 hydroxyl. The S352L structure shows that the mutation induces a dramatic 8-Å rearrangement of the catalytic loop. Because of this conformational change, Ser349 is not positioned to interact with the aldehyde substrate, conserved Glu447 is no longer poised to bind NAD(+), and Cys348 faces the wrong direction for nucleophilic attack. These structural alterations render the enzyme inactive.

Heightened stability of polcalcin Phl p 7 is correlated with strategic placement of apolar residues.

Phl p 7 exhibits atypical conformational stability and a diminutive denaturational heat capacity increment, ΔC(p). Because exposure of apolar surface largely dictates the magnitude of ΔC(p), a depressed value could signify an unusually compact unfolded state. The volume of the denatured state ensemble (DSE) is evidently inversely correlated with mean hydrophobicity [Pace et al., Protein Sci. 19 (2010) 929-943]. Interestingly, apolar residues replace more polar ones at four positions in Phl p 7. We herein examine the consequences of replacing those residues with the corresponding ones from Bra n 1, a related isoform. All four mutations - M4H, L21A, I60T, and C63A - destabilize Phl p 7. Our analysis suggests that the DSE of Phl p 7 is indeed highly compact and that the substitutions act by increasing its volume and solvent-accessibility. All four mutations increase the urea m value; L21A, I60T, and C63A also yield a perceptible increase in ΔC(p).

Crystal structure and immunogenicity of the class C acid phosphatase from Pasteurella multocida.

Pasteurella multocida is a pathogen of veterinary and medical importance. Here, we report the 1.85Å resolution crystal structure of the class C acid phosphatase from this organism (denoted rPmCCAP). The structure shows that rPmCCAP exhibits the same haloacid dehalogenase fold and dimeric assembly as the class C enzyme from Haemophilus influenzae. Formation of the dimer in solution is demonstrated using analytical ultracentrifugation. The active site is devoid of a magnesium ion due to the presence of citrate in the crystallization buffer. Absence of the metal ion minimally perturbs the active site structure, which suggests that the main role of the ion is to balance the negative charge of the substrate rather than stabilize the active site structure. The crystal lattice displays unusual crystal packing involving the C-terminal polyhistidine tag mimicking the substrate. Steady-state kinetic constants are determined for the substrates NMN, 5'-AMP, 3'-AMP, 2'-AMP, and p-nitrophenyl phosphate. The highest catalytic efficiency is observed with NMN. The production of polyclonal anti-rPmCCAP antibodies is demonstrated, and these antibodies are shown to cross-react with the H. influenzae class C phosphatase. The antibodies are used to detect PmCCAP in clinical P. multocida and Mannheimia haemolytica strains cultured from infected animals.

Solution structures of chicken parvalbumin 3 in the Ca(2+)-free and Ca(2+)-bound states.

Birds express two ß-parvalbumin isoforms, parvalbumin 3 and avian thymic hormone (ATH). Parvalbumin 3 from chicken (CPV3) is identical to rat ß-parvalbumin (ß-PV) at 75 of 108 residues. CPV3 displays intermediate Ca(2+) affinity--higher than that of rat ß-parvalbumin, but lower than that of ATH. As in rat ß-PV, the attenuation of affinity is associated primarily with the CD site (residues 41-70), rather than the EF site (residues 80-108). Structural data for rat α- and ß-parvalbumins suggest that divalent ion affinity is correlated with the similarity of the unliganded and Ca(2+)-bound conformations. We herein present a comparison of the solution structures of Ca(2+)-free and Ca(2+)-bound CPV3. Although the structures are generally similar, the conformations of residues 47 to 50 differ markedly in the two protein forms. These residues are located in the C helix, proximal to the CD binding loop. In response to Ca(2+) removal, F47 experiences much greater solvent accessibility. The side-chain of R48 assumes a position between the C and D helices, adjacent to R69. Significantly, I49 adopts an interior position in the unliganded protein that allows association with the side-chain of L50. Concomitantly, the realignment of F66 and F70 facilitates their interaction with I49 and reduces their contact with residues in the N-terminal AB domain. This reorganization of the hydrophobic core, although less profound, is nevertheless reminiscent of that observed in rat ß-PV. The results lend further support to the idea that Ca(2+) affinity correlates with the structural similarity of the apo- and bound parvalbumin conformations.

Crystal structure of a bacterial phosphoglucomutase, an enzyme involved in the virulence of multiple human pathogens.

The crystal structure of the enzyme phosphoglucomutase from Salmonella typhimurium (StPGM) is reported at 1.7 A resolution. This is the first high-resolution structural characterization of a bacterial protein from this large enzyme family, which has a central role in metabolism and is also important to bacterial virulence and infectivity. A comparison of the active site of StPGM with that of other phosphoglucomutases reveals conserved residues that are likely involved in catalysis and ligand binding for the entire enzyme family. An alternate crystal form of StPGM and normal mode analysis give insights into conformational changes of the C-terminal domain that occur upon ligand binding. A novel observation from the StPGM structure is an apparent dimer in the asymmetric unit of the crystal, mediated largely through contacts in an N-terminal helix. Analytical ultracentrifugation and small-angle X-ray scattering confirm that StPGM forms a dimer in solution. Multiple sequence alignments and phylogenetic studies show that a distinct subset of bacterial PGMs share the signature dimerization helix, while other bacterial and eukaryotic PGMs are likely monomers. These structural, biochemical, and bioinformatic studies of StPGM provide insights into the large α-D-phosphohexomutase enzyme superfamily to which it belongs, and are also relevant to the design of inhibitors specific to the bacterial PGMs.

Disparate impact of the S33V mutation on conformational stability in rat ß-parvalbumin (oncomodulin) and chicken parvalbumin 3.

Rat ß-parvalbumin (ß-PV) and chicken parvalbumin 3 (CPV3) exhibit diminished Ca(2+) affinity. Their sequences, 70% identical, are unusual in that serine replaces the consensus residue, valine, at position 33. Reasoning that the substitution of a compact, polar hydroxymethyl moiety for a bulky, apolar isopropyl group might contribute to the attenuated Ca(2+) affinities, we have characterized the S33V variants of both proteins. The impact of the mutation in CPV3 differs decidedly from that in rat ß. Whereas replacement of S33 by valine in CPV3 causes a substantial increase in the solvent-accessible apolar surface in the Ca(2+)-free protein, the mutation evidently decreases the exposed apolar surface area in rat ß. Although the mutation has a minimal effect on divalent ion affinity in both proteins, the ΔΔH and -TΔΔS changes for Ca(2+) binding in CPV3 S33V, but not rat ß S33V, are consistent with increased burial of the apolar surface. The influence of the S33V substitution on conformational stability likewise differs for rat ß-PV and CPV3. Whereas the stability of the former is virtually unperturbed by the sequence alteration, the latter is destabilized by 0.7 kcal/mol. Moreover, the mutation greatly exacerbates the tendency for CPV3 to aggregate. The concentration and scan rate dependence observed in DSC studies of CPV3 S33V denaturation suggest that unfolding proceeds through an intermediate state that is prone to aggregation. Consistent with this idea, reversible unfolding data, collected at very low protein concentration, likewise indicate that the thermal denaturation is not a two-state process.

Direct identification of the site of binding on the chaperone SecB for the amino terminus of the translocon motor SecA.

Protein export mediated by the general secretory Sec system in Escherichia coli proceeds by a dynamic transfer of a precursor polypeptide from the chaperone SecB to the SecA ATPase motor of the translocon and subsequently into and through the channel of the membrane-embedded SecYEG heterotrimer. The complex between SecA and SecB is stabilized by several separate sites of contact. Here we have demonstrated directly an interaction between the N-terminal residues 2 through 11 of SecA and the C-terminal 13 residues of SecB by isothermal titration calorimetry and analytical sedimentation velocity centrifugation. We discuss the unusual binding properties of SecA and SecB in context of a model for transfer of the precursor along the pathway of export.