Insights into the client protein release mechanism of the ATP-independent chaperone Spy.

Molecular chaperones play a central role in regulating protein homeostasis, and their active forms often contain intrinsically disordered regions (IDRs). However, how IDRs impact chaperone action remains poorly understood. Here, we discover that the disordered N terminus of the prototype chaperone Spy facilitates client release. With NMR spectroscopy and molecular dynamics simulations, we find that the N terminus can bind transiently to the client-binding cavity of Spy primarily through electrostatic interactions mediated by the N-terminal D26 residue. This intramolecular interaction results in a dynamic competition of the N terminus with the client for binding to Spy, which promotes client discharge. Our results reveal the mechanism by which Spy releases clients independent of energy input, thus enriching the current knowledge on how ATP-independent chaperones release their clients and highlighting the importance of synergy between IDRs and structural domains in regulating protein function.

Periplasmic expression of SpyTagged antibody fragments enables rapid modular antibody assembly.

Antibodies are essential tools in research and diagnostics. Although antibody fragments typically obtained from in vitro selection can be rapidly produced in bacteria, the generation of full-length antibodies or the modification of antibodies with probes is time and labor intensive. Protein ligation such as SpyTag technology could covalently attach domains and labels to antibody fragments equipped with a SpyTag. However, we found that the established periplasmic expression of antibody fragments in E. coli led to quantitative cleavage of the SpyTag by the proteases Tsp and OmpT. Here we report successful periplasmic expression of SpyTagged Fab fragments and demonstrate the coupling to separately prepared SpyCatcher modules. We used this modular toolbox of SpyCatcher proteins to generate reagents for a variety of immunoassays and measured their performance in comparison with traditional reagents. Furthermore, we demonstrate surface immobilization, high-throughput screening of antibody libraries, and rapid prototyping of antibodies based on modular antibody assembly.

Mechanism of the small ATP-independent chaperone Spy is substrate specific.

ATP-independent chaperones are usually considered to be holdases that rapidly bind to non-native states of substrate proteins and prevent their aggregation. These chaperones are thought to release their substrate proteins prior to their folding. Spy is an ATP-independent chaperone that acts as an aggregation inhibiting holdase but does so by allowing its substrate proteins to fold while they remain continuously chaperone bound, thus acting as a foldase as well. The attributes that allow such dual chaperoning behavior are unclear. Here, we used the topologically complex protein apoflavodoxin to show that the outcome of Spy's action is substrate specific and depends on its relative affinity for different folding states. Tighter binding of Spy to partially unfolded states of apoflavodoxin limits the possibility of folding while bound, converting Spy to a holdase chaperone. Our results highlight the central role of the substrate in determining the mechanism of chaperone action.

Over-activation of a nonessential bacterial protease DegP as an antibiotic strategy.

Rising antibiotic resistance urgently begs for novel targets and strategies for antibiotic discovery. Here, we report that over-activation of the periplasmic DegP protease, a member of the highly conserved HtrA family, can be a viable strategy for antibiotic development. We demonstrate that tripodal peptidyl compounds that mimic DegP-activating lipoprotein variants allosterically activate DegP and inhibit the growth of an Escherichia coli strain with a permeable outer membrane in a DegP-dependent fashion. Interestingly, these compounds inhibit bacterial growth at a temperature at which DegP is not essential for cell viability, mainly by over-proteolysis of newly synthesized proteins. Co-crystal structures show that the peptidyl arms of the compounds bind to the substrate-binding sites of DegP. Overall, our results represent an intriguing example of killing bacteria by activating a non-essential enzyme, and thus expand the scope of antibiotic targets beyond the traditional essential proteins or pathways.

IPRO+/-: Computational Protein Design Tool Allowing for Insertions and Deletions.

Insertions and deletions (indels) in protein sequences alter the residue spacing along the polypeptide backbone and consequently open up possibilities for tuning protein function in a way that is inaccessible by amino acid substitution alone. We describe an optimization-based computational protein redesign approach centered around predicting beneficial combinations of indels along with substitutions and also obtain putative substrate-docked structures for these protein variants. This modified algorithmic capability would be of interest for enzyme engineering and broadly inform other protein design tasks. We highlight this capability by (1) identifying active variants of a bacterial thioesterase enzyme ('TesA) with experimental corroboration, (2) recapitulating existing active TEM-1 ß-Lactamase sequences of different sizes, and (3) identifying shorter 4-Coumarate:CoA ligases with enhanced in vitro activities toward non-native substrates. A separate PyRosetta-based open-source tool, Indel-Maker (http://www.maranasgroup.com/software.htm), has also been created to construct computational models of user-defined protein variants with specific indels and substitutions.

A copper site is required for iron transport by the periplasmic proteins P19 and FetP.

Campylobacter jejuni is a leading cause of food-borne gastrointestinal disease in humans and uropathogenic Escherichia coli is a leading cause of urinary tract infections. Both human pathogens harbour a homologous iron uptake system (termed cjFetM-P19 in C. jejuni and ecFetM-FetP in E. coli). Although these systems are important for growth under iron limitation, the mechanisms by which these systems function during iron transport remain undefined. The copper ions bound to P19 and FetP, the homologous periplasmic proteins, are coordinated in an uncommon penta-dentate manner involving a Met-Glu-His3 motif and exhibit positional plasticity. Here we demonstrate the function of the Met and Glu residues in modulating copper binding and controlling copper positioning through site-directed variants, binding assays, and crystal structures. Growth of C. jejuni strains with these p19 variants is impaired under iron limited conditions as compared to the wild-type strain. Additionally, an acidic residue-rich secondary site is required for binding iron and function in vivo. Finally, western blot analyses demonstrate direct and specific interactions between periplasmic P19 and FetP with the large periplasmic domain of their respective inner membrane transporters cjFetM and ecFetM.

Structural Basis for the Inhibition Mechanism of Ecotin against Neutrophil Elastase by Targeting the Active Site and Secondary Binding Site.

Human neutrophil elastase (hNE) is a serine protease that plays a major role in defending the bacterial infection. However, elevated expression of hNE is reported in lung and breast cancer, among others. Moreover, hNE is a target for the treatment of cardiopulmonary diseases. Ecotin (ET) is a serine protease inhibitor present in many Gram-negative bacteria, and it plays a physiological role in inhibiting host proteases, including hNE. Despite this known interaction, the structure of the hNE-ET complex has not been reported, and the mechanism of ecotin inhibition is not available. We determined the structure of the hNE-ET complex by molecular replacement method. The structure of the hNE-ET complex revealed the presence of six interface regions comprising 50s, 60s, and 80s loops, between the ET dimer and two independent hNE monomers, which explains the high affinity of ecotin for hNE (12 pM). Notably, we observed a secondary binding site of hNE located 24 Å from the primary binding site. Comparison of the closely related trypsin-ecotin complex with our hNE-ET complex shows movement of the backbone atoms of the 80s and 50s loops by 4.6 Å, suggesting the flexibility of these loops in inhibiting a range of proteases. Through a detailed structural analysis, we demonstrate the flexibility of the hNE subsites to dock various side chains concomitant with inhibition, indicating the broad specificity of hNE against various inhibitors. These findings will aid in the design of chimeric inhibitors that target both sites of hNE and in the development of therapeutics for controlling hNE-mediated pathogenesis.

Exploring the conformational transition between the fully folded and locally unfolded substates of Escherichia coli thiol peroxidase.

Thiol peroxidase from Escherichia coli (EcTPx) is a peroxiredoxin that catalyzes the reduction of different hydroperoxides. During the catalytic cycle of EcTPx, the peroxidatic cysteine (CP) is oxidized to a sulfenic acid by peroxide, then the resolving cysteine (CR) condenses with the sulfenic acid of CP to form a disulfide bond, which is finally reduced by thioredoxin. Purified EcTPx as dithiol and disulfide behaves as a monomer under near physiological conditions. Although secondary structure rearrangements are present when comparing different redox states of the enzyme, no significant differences in unfolding free energies are observed under reducing and oxidizing conditions. A conformational change denominated fully folded (FF) to locally unfolded (LU) transition, involving a partial unfolding of αH2 and αH3, must occur to enable the formation of the disulfide bond since the catalytic cysteines are 12 Å apart in the FF conformation of EcTPx. To explore this process, the FF → LU and LU → FF transitions were studied using conventional molecular dynamics simulations and an enhanced conformational sampling technique for different oxidation and protonation states of the active site cysteine residues CP and CR. Our results suggest that the FF → LU transition has a higher associated energy barrier than the refolding LU → FF process in agreement with the relatively low experimental turnover number of EcTPx. Furthermore, in silico designed single-point mutants of αH3 enhanced locally unfolding events, suggesting that the native FF interactions in the active site are not evolutionarily optimized to fully speed-up the conformational transition of wild-type EcTPx.

The copper-responsive ScsC protein of Salmonella promotes intramacrophage survival and interacts with the arginine sensor ArtI.

The scsABCD (suppressor of copper sensitivity) locus of Salmonella encodes four proteins that resemble the disulfide folding machinery of other bacteria. Previous work has shown that Salmonella encounters toxic levels of copper during infection and the Scs system provides protection against this copper-mediated toxicity. The current work reports that expression of the soluble periplasmic protein StScsC is induced by copper and that intramacrophage survival in the presence of copper is diminished by the loss of StScsC. Using a combination of genetic and proteomic approaches, the abundance of various cysteine-containing periplasmic proteins was found to be elevated by StScsC in the Salmonella periplasm, implicating StScsC in the disulfide folding of superoxide dismutases and proteins involved in amino acid sensing and import. Co-purification and mass spectrometry approaches confirmed that the arginine-sensing periplasmic protein ArtI associates with StScsC via a disulfide interaction, and purified ArtI was shown to alter the thiol redox state of purified StScsC. This work reports the first demonstration of a redox partner for the Scs system of Salmonella and provides insights into how this bacterial pathogen responds to copper stress during infection.

Activation by substoichiometric inhibition.

Startling reports described the paradoxical triggering of the human mitogen-activated protein kinase pathway when a small-molecule inhibitor specifically inactivates the BRAF V600E protein kinase but not wt-BRAF. We performed a conceptual analysis of the general phenomenon "activation by inhibition" using bacterial and human HtrA proteases as models. Our data suggest a clear explanation that is based on the classic biochemical principles of allostery and cooperativity. Although substoichiometric occupancy of inhibitor binding sites results in partial inhibition, this effect is overrun by a concomitant activation of unliganded binding sites. Therefore, when an inhibitor of a cooperative enzyme does not reach saturating levels, a common scenario during drug administration, it may cause the contrary of the desired effect. The implications for drug development are discussed.

Subunit interactions as mediated by "non-interface" residues in living cells for multiple homo-oligomeric proteins.

Protein-protein interaction, including protein homo-oligomerization, is commonly believed to occur through a specific interface made of a limited number of amino acid residues. Here our systematic in vivo photo-crosslinking analysis via genetically incorporated unnatural amino acids unexpectedly shows that the dimerization of HdeA, an acid stress chaperone, is mediated by the residues along its whole polypeptide. These include those "forbidden" residues that are far away from the dimerization interface as judged according to the reported 3-D structure. We demonstrate that such dimerization, though intriguing, is neither a result of protein over-expression nor of any structural disturbance caused by the residue replacement. Similar unexpected dimerization also occurs for two other oligomeric proteins, IbpB (a molecular chaperone existing as polydispersed oligomers in vitro) and DegP (a protease existing as hexamers in vitro). In contrast to these three proteins, dimerization of a few other oligomeric proteins (e.g., OmpF, LamB, SurA, FtsZ and FkpA) that we similarly examined in living cells seems to be mediated only by specific residues. Together, our unexpected observations suggest that, for some oligomeric proteins such as HdeA, IbpB and DegP, their subunit interactions in living cells can also be mediated by residues other than those located at the interfaces as revealed by in vitro structure determination. Our observations might be partially explained by the formation of "encounter complex" or by protein conformational dynamics. Our findings provide new insights on understanding protein-protein interactions and encounter complex formation in living cells.

Periplasmic protein EipA determines envelope stress resistance and virulence in Brucella abortus.

Molecular components of the Brucella abortus cell envelope play a major role in its ability to infect, colonize and survive inside mammalian host cells. In this study, we have defined a role for a conserved gene of unknown function in B. abortus envelope stress resistance and infection. Expression of this gene, which we name eipA, is directly activated by the essential cell cycle regulator, CtrA. eipA encodes a soluble periplasmic protein that adopts an unusual eight-stranded ß-barrel fold. Deletion of eipA attenuates replication and survival in macrophage and mouse infection models, and results in sensitivity to treatments that compromise the cell envelope integrity. Transposon disruption of genes required for LPS O-polysaccharide biosynthesis is synthetically lethal with eipA deletion. This genetic connection between O-polysaccharide and eipA is corroborated by our discovery that eipA is essential in Brucella ovis, a naturally rough species that harbors mutations in several genes required for O-polysaccharide production. Conditional depletion of eipA expression in B. ovis results in a cell chaining phenotype, providing evidence that eipA directly or indirectly influences cell division in Brucella. We conclude that EipA is a molecular determinant of Brucella virulence that functions to maintain cell envelope integrity and influences cell division.

Identification of Nucleophilic Probes for Protease-Mediated Transpeptidation.

Proteases have evolved to mediate the hydrolysis of peptide bonds but may perform transpeptidation in the presence of a proper nucleophilic molecule that can effectively compete with water to react with the acyl-enzyme intermediate. There have been several examples of protease-mediated transpeptidation, but they are generally inefficient, and little effort has been made to systematically control the transpeptidation activity of other proteases with good nucleophiles. Here, we developed an on-bead screening approach to find a probe that functions efficiently as a nucleophile in the protease-mediated transpeptidation reaction, and we identified good probes for a model protease DegP. These probes were covalently linked to the C-termini of the cleaved peptides in a mild condition and made the selective enrichment of ligated peptides possible. We suggest that good nucleophilic probes can be found for many other proteases that act via acyl-enzyme intermediates, and these probes will help characterize their substrates.

Eukaryotic copper-only superoxide dismutases (SODs): A new class of SOD enzymes and SOD-like protein domains.

The copper-containing superoxide dismutases (SODs) represent a large family of enzymes that participate in the metabolism of reactive oxygen species by disproportionating superoxide anion radical to oxygen and hydrogen peroxide. Catalysis is driven by the redox-active copper ion, and in most cases, SODs also harbor a zinc at the active site that enhances copper catalysis and stabilizes the protein. Such bimetallic Cu,Zn-SODs are widespread, from the periplasm of bacteria to virtually every organelle in the human cell. However, a new class of copper-containing SODs has recently emerged that function without zinc. These copper-only enzymes serve as extracellular SODs in specific bacteria (i.e. Mycobacteria), throughout the fungal kingdom, and in the fungus-like oomycetes. The eukaryotic copper-only SODs are particularly unique in that they lack an electrostatic loop for substrate guidance and have an unusual open-access copper site, yet they can still react with superoxide at rates limited only by diffusion. Copper-only SOD sequences similar to those seen in fungi and oomycetes are also found in the animal kingdom, but rather than single-domain enzymes, they appear as tandem repeats in large polypeptides we refer to as CSRPs (copper-only SOD-repeat proteins). Here, we compare and contrast the Cu,Zn versus copper-only SODs and discuss the evolution of copper-only SOD protein domains in animals and fungi.

Folding while bound to chaperones.

Chaperones are important in preventing protein aggregation and aiding protein folding. How chaperones aid protein folding remains a key question in understanding their mechanism. The possibility of proteins folding while bound to chaperones was reintroduced recently with the chaperone Spy, many years after the phenomenon was first reported with the chaperones GroEL and SecB. In this review, we discuss the salient features of folding while bound in the cases for which it has been observed and speculate about its biological importance and possible occurrence in other chaperones.

Functional clues from the crystal structure of an orphan periplasmic ligand-binding protein from Treponema pallidum.

The spirochete Treponema pallidum is the causative agent of syphilis, a sexually transmitted infection of major global importance. Other closely related subspecies of Treponema also are the etiological agents of the endemic treponematoses, such as yaws, pinta, and bejel. The inability of T. pallidum and its close relatives to be cultured in vitro has prompted efforts to characterize T. pallidum's proteins structurally and biophysically, particularly those potentially relevant to treponemal membrane biology, with the goal of possibly revealing the functions of those proteins. This report describes the structure of the treponemal protein Tp0737; this polypeptide has a fold characteristic of a class of periplasmic ligand-binding proteins associated with ABC-type transporters. Although no ligand for the protein was observed in electron-density maps, and thus the nature of the native ligand remains obscure, the structural data described herein provide a foundation for further efforts to elucidate the ligand and thus the function of this protein in T. pallidum.

Structural Mimicry by a Bacterial F Box Effector Hijacks the Host Ubiquitin-Proteasome System.

Ankyrin B (AnkB/LegAU13) is a translocated F box effector essential for the intracellular replication of the pathogen Legionella pneumophila. AnkB co-opts a host ubiquitin ligase to decorate the pathogen-containing vacuole with K48-linked polyubiquitinated proteins and degrade host proteins as a source of energy. Here, we report that AnkB commandeers the host ubiquitin-proteasome system through mimicry of two eukaryotic protein domains. Using X-ray crystallography, we determined the 3D structure of AnkB in complex with Skp1, a component of the human SCF ubiquitination ligase. The structure confirms that AnkB contains an N-terminal F box similar to Skp2 and a C-terminal substrate-binding domain similar to eukaryotic ankyrin repeats. We identified crucial amino acids in the substrate-binding domain of AnkB and showed them to be essential for the function of AnkB in L. pneumophila intracellular proliferation. The study reveals how Legionella uses molecular mimicry to manipulate the host ubiquitination pathway and proliferate intracellularly.

An unusual crystal structure of ferric-enterobactin bound FepB suggests novel functions of FepB in microbial iron uptake.

Iron acquisition by siderophores is critical for the survival of most bacteria. Enterobactin is a kind of catechol siderophore that exhibits the highest affinity to iron atoms secreted by E. coli and several other species of Enterobacteriaceae. The periplasmic binding protein (PBP) FepB can transport ferric-enterobactin (Fe-Ent) from the outer membrane to the membrane-associated ATP-binding cassette transport system in E. coli. To elucidate this process, we solved the crystal structure of FepB in complex with Fe-Ent at a resolution of 1.8 Å. Consistent with previously reported NMR results, our crystal structure shows that, similar to the other type III PBPs, the FepB structure was folded with separated globular N- and C-termini linked by a long α-helix. Additionally, the structure showed that the Fe-Ent bound to the cleft between the N- and C-terminal domains. Exceptionally, FepB differs from the other known siderophore binding PBPs in that it forms a trimer by capturing four Fe-Ents that can each contribute to FepB trimerization. Dynamic light-scattering experiments are consistent with the structural observations and indicate that FepB forms a trimer in a Fe-Ent-dependent manner.

Sulfur Denitrosylation by an Engineered Trx-like DsbG Enzyme Identifies Nucleophilic Cysteine Hydrogen Bonds as Key Functional Determinant.

Exposure of bacteria to NO results in the nitrosylation of cysteine thiols in proteins and low molecular weight thiols such as GSH. The cells possess enzymatic systems that catalyze the denitrosylation of these modified sulfurs. An important player in these systems is thioredoxin (Trx), a ubiquitous, cytoplasmic oxidoreductase that can denitrosylate proteins in vivo and S-nitrosoglutathione (GSNO) in vitro However, a periplasmic or extracellular denitrosylase has not been identified, raising the question of how extracytoplasmic proteins are repaired after nitrosative damage. In this study, we tested whether DsbG and DsbC, two Trx family proteins that function in reducing pathways in the Escherichia coli periplasm, also possess denitrosylating activity. Both DsbG and DsbC are poorly reactive toward GSNO. Moreover, DsbG is unable to denitrosylate its specific substrate protein, YbiS. Remarkably, by borrowing the CGPC active site of E. coli Trx-1 in combination with a T200M point mutation, we transformed DsbG into an enzyme highly reactive toward GSNO and YbiS. The pKa of the nucleophilic cysteine, as well as the redox and thermodynamic properties of the engineered DsbG are dramatically changed and become similar to those of E. coli Trx-1. X-ray structural insights suggest that this results from a loss of two direct hydrogen bonds to the nucleophilic cysteine sulfur in the DsbG mutant. Our results highlight the plasticity of the Trx structural fold and reveal that the subtle change of the number of hydrogen bonds in the active site of Trx-like proteins is the key factor that thermodynamically controls reactivity toward nitrosylated compounds.

A molecular mechanism of chaperone-client recognition.

Molecular chaperones are essential in aiding client proteins to fold into their native structure and in maintaining cellular protein homeostasis. However, mechanistic aspects of chaperone function are still not well understood at the atomic level. We use nuclear magnetic resonance spectroscopy to elucidate the mechanism underlying client recognition by the adenosine triphosphate-independent chaperone Spy at the atomic level and derive a structural model for the chaperone-client complex. Spy interacts with its partially folded client Im7 by selective recognition of flexible, locally frustrated regions in a dynamic fashion. The interaction with Spy destabilizes a partially folded client but spatially compacts an unfolded client conformational ensemble. By increasing client backbone dynamics, the chaperone facilitates the search for the native structure. A comparison of the interaction of Im7 with two other chaperones suggests that the underlying principle of recognizing frustrated segments is of a fundamental nature.