On the unfolding of alpha-lytic protease and the role of the pro region

Molecular dynamics simulations of alpha-lytic protease (alphaLP) alone and complexed with its pro region (PRO) are performed to understand the origin of its high unfolding (and folding) barrier when it is alone and how the pro region lowers this barrier. At room temperature, alphaLP exhibits lower dynamic fluctuations than alpha-chymotrypsin. Simulation of PRO alone led to reorientation of its N terminal helix and collapse to a more compact state. A model for the uncleaved proenzyme was built and found to be stable in the time scale of the simulations. Energetic analysis suggests that the origin of strain in the uncleaved proenzyme compared with the cleaved complex is in the intramolecular backbone electrostatic interactions of the cleaved strand. In high temperature simulations, the interaction of the long beta hairpin of the enzyme with the C terminal beta sheet of PRO is among the most stable in the complex and a likely "nucleation site" for folding. In the course of unfolding, the C terminal tail of PRO is sometimes observed to intervene between the long hairpin and the aspartate loop of the enzyme, perhaps thereby lowering the energy barrier for separation of the two hairpins. Tighter interactions at the interface between the enzyme and its pro region are also occasionally observed, providing an additional mechanism for unfolding catalysis. Simulations of a mutant enzyme where the buried ion pair residues R102 and D142 were replaced by W and L, respectively, did not display any distinguishable behavior compared with the wild type.     

Correct folding of alpha-lytic protease is required for its extracellular secretion from Escherichia coli

alpha-Lytic protease is a bacterial serine protease of the trypsin family that is synthesized as a 39-kD preproenzyme (Silen, J. L., C. N. McGrath, K. R. Smith, and D. A. Agard. 1988. Gene (Amst.). 69: 237-244). The 198-amino acid mature protease is secreted into the culture medium by the native host, Lysobacter enzymogenes (Whitaker, D. R. 1970. Methods Enzymol. 19:599-613). Expression experiments in Escherichia coli revealed that the 166-amino acid pro region is transiently required either in cis (Silen, J. L., D. Frank, A. Fujishige, R. Bone, and D. A. Agard. 1989. J. Bacteriol. 171:1320-1325) or in trans (Silen, J. L., and D. A. Agard. 1989. Nature (Lond.). 341:462-464) for the proper folding and extracellular accumulation of the enzyme. The maturation process is temperature sensitive in E. coli; unprocessed precursor accumulates in the cells at temperatures above 30 degrees C (Silen, J. L., D. Frank, A. Fujishige, R. Bone, and D. A. Agard. 1989. J. Bacteriol. 171:1320-1325). Here we show that full-length precursor produced at nonpermissive temperatures is tightly associated with the E. coli outer membrane. The active site mutant Ser 195----Ala (SA195), which is incapable of self-processing, also accumulates as a precursor in the outer membrane, even when expressed at permissive temperatures. When the protease domain is expressed in the absence of the pro region, the misfolded, inactive protease also cofractionates with the outer membrane. However, when the folding requirement for either wild-type or mutant protease domains is provided by expressing the pro region in trans, both are efficiently secreted into the extracellular medium. Attempts to separate folding and secretion functions by extensive deletion mutagenesis within the pro region were unsuccessful. Taken together, these results suggest that only properly folded and processed forms of alpha-lytic protease are efficiently transported to the medium.     

alpha-lytic protease precursor: characterization of a structured folding intermediate

The bacterial alpha-lytic protease (alphaLP) is synthesized as a precursor containing a large N-terminal pro region (Pro) transiently required for correct folding of the protease [Silen, J. L., and Agard, D. A. (1989) Nature 341, 462-464]. Upon folding, the precursor is autocatalyticly cleaved to yield a tight-binding inhibitory complex of the pro region and the fully folded protease (Pro/alphaLP). An in vitro purification and refolding protocol has been developed for production of the disulfide-bonded precursor. A combination of spectroscopic approaches have been used to compare the structure and stability of the precursor with either the Pro/alphaLP complex or isolated Pro. The precursor and complex have significant similarities in secondary structure but some differences in tertiary structure, as well as a dramatic difference in stability. Correlations with isolated Pro suggest that the pro region part of the precursor is fully folded and acts to stabilize and structure the alphaLP region. Precursor folding is shown to be biphasic with the fast phase matching the rate of pro region folding. Further, the rate-limiting step in oxidative folding is formation of the disulfide bonds and autocatalytic processing occurs rapidly thereafter. These studies suggests a model in which the pro region folds first and catalyzes folding of the protease domain, forming the active site and finally causing autocatalytic cleavage of the bond separating pro region and protease. This last processing step is critical as it allows the protease N-terminus to rearrange, providing the majority of net stabilization of the product Pro/alphaLP complex.     

The pro region of BPTI facilitates folding.

The in vitro folding pathway of bovine pancreatic trypsin inhibitor (BPTI) has been described previously in terms of the disulfide-bonded intermediates that accumulate during folding of the protein. Folding is slow, occurring in hours at pH 7.3, 25 degrees C. In addition, approximately half of the BPTI molecules become trapped as a dead-end, native-like intermediate. In vivo, BPTI is synthesized as a precursor protein that includes a 13 residue amino-terminal pro region. This pro region contains a cysteine residue. We find that, in vitro, both the rate of formation and the yield of properly folded BPTI are increased substantially in a recombinant model of pro-BPTI. The cysteine residue is necessary for this effect. Moreover, a single cysteine residue, tethered to the carboxy-terminal end of BPTI with a flexible linker of repeating Ser-Gly-Gly residues, is sufficient to assist in disulfide formation. Thus, the pro region appears to facilitate folding by providing a tethered, solvent-accessible, intramolecular thiol-disulfide reagent.     

Protease pro region required for folding is a potent inhibitor of the mature enzyme.

alpha-Lytic protease, an extracellular bacterial serine protease, is synthesized with a large pro region that is required in vivo for the proper folding of the protease domain. To allow detailed mechanistic study, we have reconstituted pro region-dependent folding in vitro. The pro region promotes folding of the protease domain in the absence of other protein factors or exogenous energy sources. Surprisingly, we find that the pro region is a high affinity inhibitor of the mature protease. The pro region also inhibits the closely related Streptomyces griseus protease B, but not the more distantly related, yet structurally similar protease, elastase. Based on these data, we suggest a mechanism in which pro region binding reduces the free energy of a late folding transition state having native-like structure.     

The pro domain of beta-secretase does not confer strict zymogen-like properties but does assist proper folding of the protease domain

beta-Secretase (BACE) is a membrane-bound aspartyl protease that cleaves the amyloid precursor protein to generate the N terminus of the amyloid beta peptide. BACE is expressed as a precursor protein containing Pre, Pro, protease, transmembrane, and cytosolic domains. A soluble BACE derivative (PreProBACE460) that is truncated between the protease and transmembrane domains was produced by baculovirus-mediated expression. ProBACE460 was purified from conditioned media of infected insect cells using immobilized concanavalin A and immobilized BACE inhibitor, P10-P4' Stat(Val). Furin cleaves ProBACE460 between the Pro and protease regions to generate mature BACE460. The k(cat)/K(m) of ProBACE460 when assayed with a polypeptide substrate is only 2.3-fold less than that of BACE460. This finding and the similar inhibitory potency of P10-P4' Stat(Val) for ProBACE460 and BACE460 suggest that the Pro domain has little effect on the BACE active site. Exposure of ProBACE460 to guanidine denaturation/renaturation results in a 7-fold higher recovery of BACE activity than when BACE460 is similarly treated. The presence of free BACE Pro peptide during renaturation of BACE460 but not ProBACE460 increases recovery of activity. These findings show that the Pro domain in ProBACE460 does not suppress activity as in a strict zymogen but does appear to facilitate proper folding of an active protease domain.     

Interdependent folding of the N- and C-terminal domains defines the cooperative folding of alpha-lytic protease

Alpha-lytic protease (alphaLP) serves as an important model in achieving a quantitative and physical understanding of protein folding reactions. Synthesized as a pro-protease, alphaLP belongs to an interesting class of proteins that require pro regions to facilitate their proper folding. alphaLP's pro region (Pro) acts as a potent folding catalyst for the protease, accelerating alphaLP folding to its native conformation nearly 10(10)-fold. Structural and mutational studies suggested that Pro's considerable foldase activity is directed toward structuring the alphaLP C-terminal domain (CalphaLP), a seemingly folding-impaired domain, which is believed to contribute significantly to the high-energy folding and unfolding transition states of alphaLP. Pro-mediated nucleation of alphaLP folding within CalphaLP was hypothesized to subsequently enable the alphaLP N-terminal domain (NalphaLP) to dock and fold, completing the formation of native protease. In this paper, we find that ternary folding reactions of Pro and noncovalent NalphaLP and CalphaLP domains are unaffected by the order in which the components are added or by the relative concentrations of the alphaLP domains, indicating that neither discrete CalphaLP structuring nor docking of the two alphaLP domains is involved in the folding transition state. Instead, the rate-limiting step of these folding reactions appears to be a slow and concerted rearrangement of the NalphaLP and CalphaLP domains to form active protease. This cooperative and interdependent folding of both protease domains defines the large alphaLP folding barrier and is an apparent extension of the highly cooperative alphaLP unfolding transition that imparts the protease with remarkable kinetic stability and functional longevity.     

Two energetically disparate folding pathways of alpha-lytic protease share a single transition state

The Lysobacter enzymogenes alpha-lytic protease (alphaLP) is synthesized with a 166 amino acid pro region (Pro) that catalyzes the folding of the 198 amino acid protease into its native conformation. An extraordinary feature of this system is the very high energy barrier (DeltaG = 30 kcal mol-1) that effectively prevents alphaLP from folding in the absence of Pro (t1/2 = 1800 years). A pair of mutations has been isolated in the protease that completely suppresses the catalytic defect incurred in Pro by truncation of its last three amino acids. These mutations also accelerate the folding of alphaLP in the absence of Pro by 400-fold. An energetic analysis of the two folding reactions indicates that the mutations stabilize the transition states of both the catalyzed and uncatalyzed folding reactions by 3 kcal mol-1. This finding points to a single transition state for these two distinct and energetically disparate folding pathways, and raises the possibility that all alphaLP folding pathways share the same transition state.     

Analysis of prepro-alpha-lytic protease expression in Escherichia coli reveals that the pro region is required for activity.

The alpha-lytic protease of Lysobacter enzymogenes was successfully expressed in Escherichia coli by fusing the promoter and signal sequence of the E. coli phoA gene to the proenzyme portion of the alpha-lytic protease gene. Following induction, active enzyme was found both within cells and in the extracellular medium, where it slowly accumulated to high levels. Use of a similar gene fusion to express the protease domain alone produced inactive enzyme, indicating that the large amino-terminal pro region is necessary for activity. The implications for protein folding are discussed. Furthermore, inactivation of the protease by mutation of the catalytic serine residue resulted in the production of a higher-molecular-weight form of the alpha-lytic protease, suggesting that the enzyme is self-processing in E. coli.     

N-terminal PDZ domain is required for NHERF dimerization

NHERF, a 55 kDa PDZ-containing protein, binds receptors and ion transporters to mediate signal transduction at the plasma membrane. Recombinant NHERF demonstrated an apparent size of 150 kDa on gel filtration, which could be reduced to approximately 55 kDa by protein denaturing agents, consistent with the formation of NHERF dimers. Biosensor studies established the time- and concentration-dependent dimerization of NHERF. Overlays of recombinant NHERF fragments suggested that NHERF dimerization was principally mediated by the N-terminal PDZ-I domain. In PS120 cells, reversible protein phosphorylation modulated NHERF dimerization and suggested a role for NHERF dimers in hormonal signaling.     

A C-terminal region of signal peptide peptidase defines a functional domain for intramembrane aspartic protease catalysis

Intramembrane proteolysis is now firmly established as a prominent biological process, and structure elucidation is emerging as the new frontier in the understanding of these novel membrane-embedded enzymes. Reproducing this unusual hydrolysis within otherwise water-excluding transmembrane regions with purified proteins is a challenging prerequisite for such structural studies. Here we show the bacterial expression, purification, and reconstitution of proteolytically active signal peptide peptidase (SPP), a membrane-embedded enzyme in the presenilin family of aspartyl proteases. This finding formally proves that, unlike presenilin, SPP does not require any additional proteins for proteolysis. Surprisingly, the conserved C-terminal half of SPP is sufficient for proteolytic activity; purification and reconstitution of this engineered fragment of several SPP orthologues revealed that this region defines a functional domain for an intramembrane aspartyl protease. The discovery of minimal requirements for intramembrane proteolysis should facilitate mechanistic and structural analysis and help define general biochemical principles of hydrolysis in a hydrophobic environment.     

The N-terminal extracellular domain is required for polycystin-1-dependent channel activity

Autosomal dominant polycystic kidney disease (PKD) is caused by mutation of polycystin-1 or polycystin-2. Polycystin-2 is a Ca(2+)-permeable cation channel. Polycystin-1 is an integral membrane protein of less defined function. The N-terminal extracellular region of polycystin-1 contains potential motifs for protein and carbohydrate interaction. We now report that expression of polycystin-1 alone in Chinese hamster ovary (CHO) cells and in PKD2-null cells can confer Ca(2+)-permeable non-selective cation currents. Co-expression of a loss-of-function mutant of polycystin-2 in CHO cells does not reduce polycystin-1-dependent channel activity. A polycystin-1 mutant lacking approximately 2900 amino acids of the extracellular region is targeted to the cell surface but does not produce current. Extracellular application of antibodies against the immunoglobulin-like PKD domains reduces polycystin-1-dependent current. These results support the hypothesis that polycystin-1 is a surface membrane receptor that transduces the signal via changes in ionic currents.     

pH-dependent interactions and the stability and folding kinetics of the N-terminal domain of L9. Electrostatic interactions are only weakly formed in the transition state for folding

The role of electrostatic interactions in the stability and the folding of the N-terminal domain of the ribosomal protein L9 (NTL9) was investigated by determining the effects of varying the pH conditions. Urea denaturations and thermal unfolding experiments were used to measure the free energy of folding, DeltaG degrees, at 18 different pH values, ranging from pH 1.1 to pH 10.5. Folding rates were measured at 19 pH values between pH 2.1 and pH 9.5, and unfolding rates were determined at 15 pH values in this range using stopped-flow fluorescence experiments. The protein is maximally stable between pH 5.5 and 7.5 with a value of DeltaG degrees =4.45 kcal mol(-1). The folding rate reaches a maximum at pH 5.5, however the change in folding rates with pH is relatively modest. Over the pH range of 2.1 to 5.5 there is a small increase in folding rates, ln (k(f)) changes from 5.1 to 6.8. However, the change in stability is more dramatic, with a difference of 2.6 kcal mol(-1) between pH 2.0 and pH 5.4. The change in stability is largely due to the smaller barrier for unfolding at low pH values. The natural log of the unfolding rates varies by approximately four units between pH 2.1 and pH 5.5. The stability of the protein decreases above pH 7.5 and again the change is largely due to changes in the unfolding rate. ln (k(f)) varies by less than one unit between pH 5.5 and pH 9.5 while DeltaG degrees decreases by 2.4 kcal mol(-1) over the range of pH 5. 4 to pH 10.0, which corresponds to a change in ln K(eq) of 4.0. These studies show that pH-dependent interactions contribute significantly to the overall stability of the protein but have only a small effect upon the folding kinetics, indicating that electrostatic interactions are weakly formed in the transition state for folding.     

A C-terminal domain conserved in precursor processing proteases is required for intramolecular N-terminal maturation of pro-Kex2 protease.

The Kex2 protease of the yeast Saccharomyces cerevisiae is the prototype of a family of eukaryotic subtilisin homologs thought to process prohormones and other precursors in the secretory pathway. Deletion analysis of Kex2 protease shows that a sequence of 154-159 residues carboxyl to the subtilisin domain is essential for the formation of active enzyme. Disruption of this region, termed the 'P-domain', blocks the normally rapid intra-molecular cleavage of the N-terminal pro-segment of pro-Kex2 protease in the endoplasmic reticulum (ER). The C-terminal boundary of the P-domain coincides closely with the endpoint of similarity between Kex2 protease and its mammalian homologues. The conservation of and functional requirement for the P-domain sharpens the distinction between a 'Kex2 family' of processing enzymes and degradative 'subtilases', and implies that the Kex2-related enzymes have in common entirely novel structural features that are important in the maturation of precursor polypeptide substrates. Failure to cleave the N-terminal pro-domain, due either to truncation of the P-domain or to mutation of the active site histidine or serine, results in stable, intracellular retention of pro-enzyme, apparently in the ER. Thus pro-Kex2 protease appears to contain an ER retention signal which is removed or destroyed by cleavage of the pro-domain.     

The basic region of the diaphanous-autoregulatory domain (DAD) is required for autoregulatory interactions with the diaphanous-related formin inhibitory domain

Mammalian diaphanous-related (mDia) formins act as Rho GTPase effectors during cytoskeletal remodeling. Rho binding to mDia amino-terminal GTPase-binding domains (GBDs) causes the adjacent Dia-inhibitory domain (DID) to release the carboxyl-terminal Dia-autoregulatory (DAD) domain that flanks the formin homology-2 (FH2) domain. The release of DAD allows the FH2 domain to then nucleate and elongate nonbranched actin filaments. DAD, initially discovered as a region of homology shared between a phylogenetically divergent set of formin proteins, is comprised of a core motif, MDXLLXL, and an adjacent region is comprised of numerous basic residues, typically RRKR in the mDia family. Here, we show that these specific amino acids within the basic region of DAD contribute to the binding of DID and therefore the maintenance of the mDia autoregulatory mechanism. In addition, expression of full-length versions of mDia2 containing amino acid substitutions in either the DAD core or basic regions causes profound changes in the F-actin architecture, including the formation of filopodia-like structures that rapidly elongate from the cell edge. These studies further refine our understanding of the molecular contribution of DAD to mDia control and the role of mDia2 in the assembly of membrane protrusions.     

The N-terminal pro region mediates retention of unprocessed type-I PME in the Golgi apparatus

The pectin matrix of the cell wall, a complex and dynamic network, impacts on cell growth, cell shape and signaling processes. A hallmark of pectin structure is the methylesterification status of its major component, homogalacturonan (HGA), which affects the biophysical properties and enzymatic turnover of pectin. The pectin methylesterases (PMEs), responsible for de-esterification, encompass a protein family of more than 60 isoforms in the Arabidopsis genome. The pivotal role of PME in the regulation of pectin properties also requires tight control at the post-translational level. Type-I PMEs are characterized by an N-terminal pro region, which exhibits homology with pectin methylesterase inhibitors (PMEIs). Here, we demonstrate that the proteolytic removal of the N-terminal pro region depends on conserved basic tetrad motifs, occurs in the early secretory pathway, and is required for the subsequent export of the PME core domain to the cell wall. In addition, we demonstrate the involvement of AtS1P, a subtilisin-like protease, in Arabidopsis PME processing. Our results indicate that the pro region operates as an effective retention mechanism, keeping unprocessed PME in the Golgi apparatus. Consequently, pro-protein processing could constitute a post-translational mechanism regulating PME activity.     

The N-terminal cytokine binding domain of LIFR is required for CNTF binding and signaling

Ciliary neurotrophic factor (CNTF) forms a functional receptor complex containing the CNTF receptor, gp130, and the leukemia inhibitory factor receptor (LIFR). However, the nature and stoichiometry of the receptor-mediated interactions in this complex have not yet been fully resolved. We show here that signaling by CNTF, but not by LIF or oncostatin M (OSM), was abolished in cells overexpressing a LIFR mutant with the N-terminal cytokine binding domain deleted. Our results illustrate molecular differences between the CNTF active receptor complex and those of LIF and OSM and provide further support for the hexameric model of the CNTF receptor complex.     

The folding landscape of an alpha-lytic protease variant reveals the role of a conserved beta-hairpin in the development of kinetic stability

Most secreted bacterial proteases, including alpha-lytic protease (alphaLP), are synthesized with covalently attached pro regions necessary for their folding. The alphaLP folding landscape revealed that its pro region, a potent folding catalyst, is required to circumvent an extremely large folding free energy of activation that appears to be a consequence of its unique unfolding transition. Remarkably, the alphaLP native state is thermodynamically unstable; a large unfolding free energy barrier is solely responsible for the persistence of its native state. Although alphaLP folding is well characterized, the structural origins of its remarkable folding mechanism remain unclear. A conserved beta-hairpin in the C-terminal domain was identified as a structural element whose formation and positioning may contribute to the large folding free energy barrier. In this article, we characterize the folding of an alphaLP variant with a more favorable beta-hairpin turn conformation (alphaLP(beta-turn)). Indeed, alphaLP(beta-turn) pro region-catalyzed folding is faster than that for alphaLP. However, instead of accelerating spontaneous folding, alphaLP(beta-turn) actually unfolds more slowly than alphaLP. Our data support a model where the beta-hairpin is formed early, but its packing with a loop in the N-terminal domain happens late in the folding reaction. This tight packing at the domain interface enhances the kinetic stability of alphaLP(beta-turn), to nearly the same degree as the change between alphaLP and a faster folding homolog. However, alphaLP(beta-turn) has impaired proteolytic activity that negates the beneficial folding properties of this variant. This study demonstrates the evolutionary limitations imposed by the simultaneous optimization of folding and functional properties.