The Autocatalytic Cleavage Domain Is Not Required for the Activity of ScpC, a Virulence Protease from Streptococcus pyogenes: A Structural Insight.

Group A Streptococcus (GAS, or Streptococcus pyogenes) is a leading human bacterial pathogen with diverse clinical manifestations, ranging from mild to life-threatening and to severe immune sequela. These diseases, combined, account for more than half a million deaths per year, globally. To accomplish its vast pathogenic potential, GAS expresses a multitude of virulent proteins, including the pivotal virulence factor ScpC. ScpC is a narrow-range surface-exposed subtilisin-like serine protease that cleaves the last 14 C-terminal amino acids of interleukin 8 (IL-8 or CXCL8) and impairs essential IL-8 signaling processes. As a result, neutrophil migration, bacterial killing, and the formation of neutrophil extracellular traps are strongly impaired. Also, ScpC has been identified as a potential vaccine candidate. ScpC undergoes an autocatalytic cleavage between Gln244 and Ser245, resulting in two polypeptide chains that assemble together forming the active protease. Previously, we reported that the region harboring the autocatalytic cleavage site, stretching from Gln213 to Asp272, is completely disordered. Here, we show that a deletion mutant (ScpCΔ60) of this region forms a single polypeptide chain, whose crystal structure we determined at 2.9 Å resolution. Moreover, we show that ScpCΔ60 is an active protease capable of cleaving its substrate IL-8 in a manner comparable to that of the wild type. These studies improve our understanding of the proteolytic activity of ScpC.

NMR structure of CmPI-II, a non-classical Kazal protease inhibitor: Understanding its conformational dynamics and subtilisin A inhibition.

Subtilisin-like proteases play crucial roles in host-pathogen interactions. Thus, protease inhibitors constitute important tools in the regulation of this interaction. CmPI-II is a Kazal proteinase inhibitor isolated from Cenchritis muricatus that inhibits subtilisin A, trypsin and elastases. Based on sequence analysis it defines a new group of non-classical Kazal inhibitors. Lacking solved 3D structures from this group prevents the straightforward structural comparison with other Kazal inhibitors. The 3D structure of CmPI-II, solved in this work using NMR techniques, shows the typical fold of Kazal inhibitors, but has significant differences in its N-terminal moiety, the disposition of the CysI-CysV disulfide bond and the reactive site loop (RSL) conformation. The high flexibility of its N-terminal region, the RSL, and the α-helix observed in NMR experiments and molecular dynamics simulations, suggest a coupled motion of these regions that could explain CmPI-II broad specificity. The 3D structure of the CmPI-II/subtilisin A complex, obtained by modeling, allows understanding of the energetic basis of the subtilisin A inhibition. The residues at the P2 and P2' positions of the inhibitor RSL were predicted to be major contributors to the binding free energy of the complex, rather than those at the P1 position. Site directed mutagenesis experiments confirmed the Trp14 (P2') contribution to CmPI-II/subtilisin A complex formation. Overall, this work provides the structural determinants for the subtilisin A inhibition by CmPI-II and allows the designing of more specific and potent molecules. In addition, the 3D structure obtained supports the existence of a new group in non-classical Kazal inhibitors.

Soft interactions at nanoparticles alter protein function and conformation in a size dependent manner.

Weak protein-nanoparticle (NP) interactions are studied in a low binding regime as a model for the soft protein corona around nanoparticles in complex biological fluids. Noncovalent, reversible interactions between Subtilisin Carlsberg (SC) and silica NPs shows significant alteration in conformation and enzymatic activity in a NP-size dependent manner. Very weak interactions between SC and silica NPs were revealed by centrifugation-based separations and further supported by small-angle X-ray scattering, while bovine serum albumin was used as a strongly interacting reference. Secondary and tertiary structure changes of SC were studied via circular dichroism and correlated to enzymatic activity where the enzyme kinetics showed a critical role for nanoparticle size.

Insights from bacterial subtilases into the mechanisms of intramolecular chaperone-mediated activation of furin.

Prokaryotic subtilisins and eukaryotic proprotein convertases (PCs) are two homologous protease subfamilies that belong to the larger ubiquitous super-family called subtilases. Members of the subtilase super-family are produced as zymogens wherein their propeptide domains function as dedicated intramolecular chaperones (IMCs) that facilitate correct folding and regulate precise activation of their cognate catalytic domains. The molecular and cellular determinants that modulate IMC-dependent folding and activation of PCs are poorly understood. In this chapter we review what we have learned from the folding and activation of prokaryotic subtilisin, discuss how this has molded our understanding of furin maturation, and foray into the concept of pH sensors, which may represent a paradigm that PCs (and possibly other IMC-dependent eukaryotic proteins) follow for regulating their biological functions using the pH gradient in the secretory pathway.

Effect of PEG modification on subtilisin Carlsberg activity, enantioselectivity, and structural dynamics in 1,4-dioxane.

The employment of enzymes as catalysts within organic media has traditionally been hampered by the reduced enzymatic activities when compared to catalysis in aqueous solution. Although several complementary hypotheses have provided mechanistic insights into the causes of diminished activity, further development of biocatalysts would greatly benefit from effective chemical strategies (e.g., PEGylation) to ameliorate this event. Herein we explore the effects of altering the solvent composition from aqueous buffer to 1,4-dioxane on structural, dynamical, and catalytic properties of the model enzyme subtilisin Carlsberg (SBc). Furthermore, we also investigate the effects of dissolving the enzyme in 1,4-dioxane through chemical modification with poly(ethylene)-glycol (PEG, M(W) = 20 kDa) on these enzyme properties. In 1,4-dioxane a 10(4)-fold decrease in the enzyme's catalytic activity was observed for the hydrolysis reaction of vinyl butyrate with D(2)O and a 50% decrease in enzyme structural dynamics as evidenced by reduced amide H/D exchange kinetics occurred. Attaching increasing amounts of PEG to the enzyme reversed some of the activity loss. Evaluation of the structural dynamic behavior of the PEGylated enzyme within the organic solvent revealed an increase in structural dynamics at increased PEGylation. Correlation analysis between the catalytic and structural dynamic parameters revealed that the enzyme's catalytic activity and enantioselectivity depended on the changes in protein structural dynamics within 1,4-dioxane. These results demonstrate the importance of protein structural dynamics towards regulating the catalytic behavior of enzymes within organic media.

Comparison of theoretical and experimental data to evaluate substrate diffusional limitations for crown ether- and methyl-beta-cyclodextrin-activated serine protease subtilisin Carlsberg in tetrahydrofuran.

Simple co-lyophilization of serine protease subtilisin Carlsberg with [12]-crown ether-4 (12-crown-4) or methyl-beta-cyclodextrin (MbetaCD) drastically increases its catalytic activity in organic solvents. We investigated whether the improved activity would cause substrate diffusional limitations. To experimentally assess the issue, the enzyme was inactivated with PMSF. Different amounts of active and inactive subtilisin were codissolved in 10 mM phosphate buffer (pH 7.8) followed by lyophilization with or without 12-crown-4 or MbetaCD. Initial rates for the transesterification reaction of N-acetyl-L-phenylalanine ethyl ester and 1-propanol in anhydrous THF were plotted vs. the amount of active enzyme present in the formulations. For all three enzyme formulations a linear relationship was observed and the results clearly show that activation of subtilisin Carlsberg by crown ethers and MbetaCD did not cause diffusional limitations. This was somewhat surprising because theoretical models predicted such diffusional limitations for the activated formulations. However, investigation of the protein powder particles obtained after co-lyophilization with 12-crown-4 and MbetaCD revealed a drastically reduced particle size for these formulations when suspended in THF. The particle micronization afforded by the excipients prevented substrate diffusional limitations, a factor that should be taken into account when designing improved enzyme formulations for synthetic applications in organic solvents.

Furin: a mammalian subtilisin/Kex2p-like endoprotease involved in processing of a wide variety of precursor proteins.

Limited endoproteolysis of inactive precursor proteins at sites marked by paired or multiple basic amino acids is a widespread process by which biologically active peptides and proteins are produced within the secretory pathway in eukaryotic cells. The identification of a novel family of endoproteases homologous with bacterial subtilisins and yeast Kex2p has accelerated progress in understanding the complex mechanisms underlying the production of the bioactive materials. Seven distinct proprotein convertases of this family (furin, PC2, PC1/PC3, PC4, PACE4, PC5/PC6, LPC/PC7/PC8/SPC7) have been identified in mammalian species, some having isoforms generated via alternative splicing. The family has been shown to be responsible for conversion of precursors of peptide hormones, neuropeptides, and many other proteins into their biologically active forms. Furin, the first proprotein convertase to be identified, has been most extensively studied. It has been shown to be expressed in all tissues and cell lines examined and to be mainly localized in the trans-Golgi network, although some proportion of the furin molecules cycle between this compartment and the cell surface. This endoprotease is capable of cleaving precursors of a wide variety of proteins, including growth factors, serum proteins, including proteases of the blood-clotting and complement systems, matrix metalloproteinases, receptors, viral-envelope glycoproteins and bacterial exotoxins, typically at sites marked by the consensus Arg-Xaa-(Lys/Arg)-Arg sequence. The present review covers the structure and function of mammalian subtilisin/Kex2p-like proprotein convertases, focusing on furin (EC 3.4.21.85).

Folding pathway mediated by an intramolecular chaperone: characterization of the structural changes in pro-subtilisin E coincident with autoprocessing.

Mechanisms by which many N-terminal propeptides facilitate folding of proteins are unknown. The maturation of such proteins from their precursors involve three steps, namely: (1) folding of the precursor, (2) autoprocessing of the propeptide from the N terminus and (3) degradation of the cleaved propeptide. Using subtilisin E we have analyzed the mechanism of propeptide-mediated protein folding. Two active site mutations allow us to trap intermediates at stages of autoprocessing and degradation. An analysis of these intermediates has shown the existence of a molten-globule-like intermediate on the folding pathway. After autoprocessing of the propeptide, this intermediate undergoes a structural reorganization which reduces solvent-accessible hydrophobic surface area and increases the amount of its tertiary structure. Removal of the propeptide from the mature enzyme in this intermediate state occurs only by proteolytic degradation and contributes to the stability of the active enzyme.

1H NMR spectroscopic studies of selenosubtilisin.

Anomalously low-field signals in 1H NMR spectra of serine proteases provide valuable information on the protonation state of the catalytic histidine residue. We have examined the pH dependence of the deshielded protons of three different oxidation states of selenosubtilisin, a semisynthetic selenoenzyme with significant peroxidase activity, in order to evaluate the influence of the selenium prosthetic group on the hydrogen-bonding network in the modified active site. In the spectra of the anionic seleninate and selenolate derivatives, two resonances were observed at 18.0 and 15.5/14.0 ppm, assigned respectively to the N delta 1 and N epsilon 2 protons of protonated His64. These signals were apparent from pH 4 to above pH 10, indicating that the negatively charged prosthetic group increases the stability of the imidazolium dramatically, raising its pKa by at least 3-4 pH units. In contrast, a neutral selenenyl sulfide species exhibits no deshielded proton signals at 18 ppm at any pH but has a weak signal at 14.1 ppm above pH 7 which was assigned to the N delta 1 imidazole proton of neutral His64. While the pKa of His64 appears normal (approximately 7) in this derivative, the selenenyl sulfide substitution may alter the orientation of the imidazole ring within the active site for steric reasons. Together with data on the influence of pH on peroxidase activity, these results suggest that selenosubtilisin's His64 acts as a general acid facilitating the reduction of the selenenyl sulfide to selenolate by thiols.

Neutron structure of subtilisin BPN': effects of chemical environment on hydrogen-bonding geometries and the pattern of hydrogen-deuterium exchange in secondary structure elements.

The neutron structure of subtilisin BPN' has been refined and analyzed at 2.0-A resolution. The structure studied was a mutant variant of subtilisin, Met222----Gln, and was used because large, uninhibited crystals could be grown, which was not the case for the native molecule. Comparison of the structure with that of the native molecule indicated that the two structures are essentially the same. Using the capability of the neutron method to locate hydrogen and deuterium atoms, the protonation states of the six histidine residues were assigned. The active site histidine, His64, was found to be neutral at the pH of the analysis (pH 6.1). This group has an unexpectedly low pKa compared to assignments made by other techniques. The altered pKa of the group could result from electrostatic effects of other molecules in the crystal lattice. The dihedral conformations of a majority of the hydroxyl rotors were assigned. The preferred orientation was trans (180 degrees) with the other two low-energy conformers (60 degrees, 300 degrees) about equally populated. For the serines, about 21% of the hydroxyls act exclusively as H-bond acceptors and 37% as H-bond donors, and in 42% the group functions as both. The experimentally observed dihedral conformations were compared to predicted conformations based on calculated energy criteria and showed a strong correspondence. Deviation from low-energy states could usually be explained by local electrostatic effects. The hydrogen exchange pattern of subtilisin identified the beta-sheet and alpha-helix secondary structure elements to be the most resistant to exchange. Fifty-five percent of the peptide amide hydrogens were fully exchanged, 15% unexchanged, and 30% partially exchanged. The largest concentration of unexchanged sites was in the seven-stranded parallel beta-sheet, in which there were 11 fully protected groups. Little correlation was found between H-bond length and angle and a peptide group's susceptibility toward exchange. Of the five alpha-helices the most protected from exchange is the one defined by residues 224-236. The pattern of exchange identifies regions in this helix where the H-bonding regularity is disrupted.

Refined crystal structures of subtilisin novo in complex with wild-type and two mutant eglins. Comparison with other serine proteinase inhibitor complexes.

The crystal structures of the complexes formed between subtilisin Novo and three inhibitors, eglin c, Arg45-eglin c and Lys53-eglin c have been determined using molecular replacement and difference Fourier techniques and refined at 2.4 A, 2.1 A, and 2.4 A resolution, respectively. The mutants Arg45-eglin c and Lys53-eglin c were constructed by site-directed mutagenesis in order to investigate the inhibitory specificity and stability of eglin c. Arg45-eglin became a potent trypsin inhibitor, in contrast to native eglin, which is an elastase inhibitor. This specificity change was rationalized by comparing the structures of Arg45-eglin and basic pancreatic trypsin inhibitor and their interactions with trypsin. The residue Arg53, which participates in a complex network of hydrogen bonds formed between the core and the binding loop of eglin c, was replaced with the shorter basic amino acid lysine in the mutant Lys53-eglin. Two hydrogen bonds with Thr44, located in the binding loop, can no longer be formed but are partially restored by a water molecule bound in the vicinity of Lys53. Eglin c in complexes with both subtilisin Novo and subtilisin Carlsberg was crystallized in two different space groups. Comparison of the complexes showed a rigid body rotation for the eglin c core of 11.5 degrees with respect to the enzyme, probably caused by different intermolecular contacts in both crystal forms.