Crystal structure of a mycobacterial secretory protein Rv0398c and in silico prediction of its export pathway.

Secretory proteins are used by pathogenic bacteria to manipulate the host systems and compete with other microorganisms, thereby enabling their survival in their host. Similar to other bacteria, secretory proteins of Mycobacterium tuberculosis also play a pivotal role in evading immune response within hosts, thereby leading to acute and latent tuberculosis infection. Prokaryotes have several classes of bacterial secretory systems out of which the Sec and Tat pathways are the most conserved in Mtb to transport proteins across the cytoplasmic membrane. Here, we report the crystal structure of a secretory protein, Rv0398c determined to 1.9 Å resolution. The protein comprises a core of antiparallel ß sheets surrounded by α helices adopting a unique ß sandwich fold. Structural comparison with other secretory proteins in Mtb and other pathogenic bacteria reveals that Rv0398c may be secreted via the Sec pathway. Our structural and in silico analyses thus provide mechanistic insights into the pathway adopted by Mtb to transport out secretory protein, Rv0398c which will facilitate the invasion to the host immune system.

Insight into the Lysozyme-Induced Aggregation of Aromatic Amino Acid-Functionalized Gold Nanoparticles: Impact of the Protein Conjugation and Lipid Corona on the Aggregation Phenomena.

The aggregation and subsequent precipitation of gold nanoparticles (Au NPs) in the presence of protein molecules restrict the usefulness of NPs in biomedical applications. Till now, the influence of different properties of Au NPs (size, surface charge, surface coatings) and proteins (surface charge, chemical modification, folded and unfolded states) and pH and ionic strength of the solution on the aggregation of both Au NPs and proteins has been thoroughly discussed in the literature. However, the underlying different mechanistic pathways of the protein concentration-dependent aggregation of both Au NPs and proteins are poorly understood. The impact of the lipid corona on the protein-induced Au NP aggregation has remained an unresolved issue. In this context, we investigate the interaction of the negatively charged aromatic amino acid (phenylalanine and tyrosine)-functionalized gold nanoparticles (Au-AA NPs) with the positively charged globular protein lysozyme at different protein concentrations and compare the results with those of conventional citrate-functionalized Au NPs (Au-Cit NPs). Next, we conjugate lipids and proteins to Au NPs to impede the aggregation of Au NPs induced by the lysozyme. Our results reveal that the aggregation mechanism of the Au-AA NPs is distinctly different at low and high protein concentrations with the uniqueness of the Au-AA NPs over the Au-Cit NPs. Furthermore, we find that human serum albumin (HSA) protein-conjugated Au-AA and Au-Cit NPs are more effective in preventing the lysozyme-induced Au NP aggregation than bovine serum albumin (BSA)-conjugated Au NPs. For the first time, we also report the significant role of "hard" and "soft" lipid coronas in the aggregation of amino acid (phenylalanine)-functionalized gold nanoparticles in the presence of lysozyme protein.

Enhancement of angiogenin inhibition by polyphenol-capped gold nanoparticles.

Angiogenin (Ang), is a ribonucleolytic protein that is associated with angiogenesis, the formation of blood vessels. The involvement of Ang in vascularisation makes it a potential target for the identification of compounds that have the potential to inhibit the process. The compounds may be assessed for their ability to inhibit the ribonucleolytic activity of the protein and subsequently blood vessel formation, a crucial requirement for tumor formation. We report an inhibition of the ribonucleolytic activity of Ang with the gallate containing green tea polyphenols, ECG and EGCG that exhibits an increased efficacy upon forming polyphenol-capped gold nanoparticles (ECG-AuNPs and EGCG-AuNPs). The extent of inhibition was confirmed using an agarose gel-based assay followed by fluorescence titration studies that indicated a hundred fold stronger binding of polyphenol-capped gold nanoparticles (GTP-AuNPs) compared to the bare polyphenols. Interestingly, we found a change in the mode of inhibition from a noncompetitive type to a competitive mode of inhibition in case of the GTP-AuNPs, which is in agreement with the 'n' values obtained from the fluorescence quenching studies. The effect on angiogenesis has also been assessed by the chorioallantoic membrane (CAM) assay. We find an increase in the inhibition potency of GTP-AuNPs that could find applications in the development of anti-angiogenic compounds.

Underlying Mechanisms for the Modulation of Self-Assembly and the Intrinsic Fluorescent Properties of Amino Acid-Functionalized Gold Nanoparticles.

The origin of the blue fluorescence of proteins and peptides in the visible region has been a subject of intense debate despite several efforts. Although aromatic amino acids, namely tryptophan (Trp), tyrosine (Tyr), and phenylalanine (Phe) are responsible for the intrinsic luminescence of proteins and peptides, the underlying mechanism and contributions of these amino acids to the unusual blue fluorescence are still not well resolved. In the present endeavor, we show that the clusterization of both aromatic and aliphatic amino acids on the surface of the gold nanoparticles (Au NPs) leads to clusteroluminescence, which could be linked to the unusual fluorescence properties of the proteins and peptides and have been ignored in the past. The amino acid monomers initially form small aggregates through clusterization, which provides the fundamental building blocks to establish the amyloid structure as well as the luminescence property. Because of the clusterization, these Au NPs/nano-aggregate systems are also found to exhibit remarkable stability against the freeze-thaw cycle and several other external stimuli, which can be useful for biological and biomedical applications.

Structural, Dynamic, and Functional Characterization of a DnaX Mini-intein Derived from Spirulina platensis Provides Important Insights into Intein-Mediated Catalysis of Protein Splicing.

Protein splicing is a self-catalyzed post-translational modification in which the intein enzyme excises itself from a precursor protein and ligates the flanking sequences to produce a mature protein. We report the solution structure of a 136-residue DnaX mini-intein enzyme derived from the cyanobacterium Spirulina platensis. This sequence adopts a well-defined globular structure and forms a horseshoe-shaped fold commonly found in the HINT (hedgehog intein) topology. Backbone dynamics and hydrogen exchange experiments revealed conserved motions on various time scales, which is proposed to be a characteristic of the intein fold. Interestingly, several dynamic motions were found in symmetrically equivalent positions within the protein structure, which might be a consequence of the symmetrical intein fold. In cell splicing activity showed that Spl DnaX mini-intein is a highly active enzyme. The precursor protein was not detected at any timepoint of the assay. Apart from the splicing reaction, catalytic cleavage at the N- and C-termini of the precursor protein was also observed. To determine the roles of the catalytic residues in splicing and cleavage reactions, all combinations of alanine mutations of these residues were generated and functionally characterized. This in-depth analysis revealed cooperativity between these catalytic residues, which suppresses the N- and C-terminal cleavage reactions and enhances the yield of the spliced product. Overall, this study provides a thorough structural, dynamic, and functional characterization of a new intein sequence and adds to the collection of these unique enzymes that have found tremendous applications in biochemistry and biotechnology.

Interaction of aliphatic amino acids with zwitterionic and charged lipid membranes: hydration and dehydration phenomena.

In the present contribution, we investigate the interactions of lipid bilayer membranes of different charges and different phase states with aliphatic amino acids of varying charge (aspartic acid, glutamic acid, arginine and lysine) and hydrophobicity (serine, leucine and valine) by steady state and time-resolved spectroscopic techniques, dynamic light scattering (DLS) measurements and confocal imaging (CLSM). The study reveals that negatively charged amino acids such as aspartic acid and glutamic acid interact strongly with the lipid membranes particularly with negatively charged lipid membranes by stabilizing their gel phase. On the other hand, positively charged amino acids bring in hydration in the membranes. We explain this unique observation by the shift in pKa of amino acids in the vicinity of the lipid membranes and solvation and desolvation processes in the light of recent computer simulations. We also find that hydrogen bonding plays a significant role in governing the interaction of aliphatic amino acids with zwitterionic lipid membranes. The more polar serine bearing a hydroxyl group at the terminal carbon offers a stronger interaction with the lipid bilayer membranes as compared to its analogues leucine and valine, which are hydrophobic in nature.

Influence of Trivalent Metal Ions on Lipid Vesicles: Gelation and Fusion Phenomena.

In this contribution, we report the interaction of 1,2-dimyristoyl- sn-glycero-3-phosphocholine (DMPC) lipid vesicles with a series of trivalent metal ions of the same group, namely, Al3+, Ga3+, and In3+, to get a distinct view of the effect of size, effective charge, and hydration free energy of these metal ions on lipid vesicles. We employed steady-state and time-resolved spectroscopic techniques including time-resolved anisotropy measurement, confocal imaging, and dynamic light scattering (DLS) measurement to probe the interaction. Our study reveals that all of the three trivalent metal ions induce gelation in lipid vesicles by removing water molecules from the interfacial region. The extent of gelation induced by the metal ions follows the order of In3+ > Ga3+ ≥ Al3+. We explain this observation in light of different free-energy terms. Notably, the degree of interaction for trivalent metal ions is higher as compared to that for divalent metal ions at physiological pH (pH ∼ 7.0). Most importantly, we observe that unlike divalent metal ions, trivalent metal ions dehydrate the lipid vesicles even at lower pH. The DLS measurement and confocal imaging indicate that In3+ causes significant aggregation or fusion of the PC vesicles, while Al3+ and Ga3+ did not induce any aggregation at the experimental concentration. We employ Derjaguin-Landau-Vervey-Overbeek (DLVO) theory to explain the aggregation phenomena induced by In3+.

Effect of Surface Ligand and Temperature on Lipid Vesicle-Gold Nanoparticle Interaction: A Spectroscopic Investigation.

We herein investigate the interactions of differently functionalized anionic and cationic gold nanoparticles (AuNPs) with zwitterionic phosphocholine (PC) as well as inverse phosphocholine (iPC) lipid bilayers via spectroscopic measures. In this study, we used PC lipids with varying phase-transition temperatures, i.e., DMPC ( Tm = 24 °C), DOPC ( Tm = -20 °C), and iPC lipid DOCP ( Tm = -20 °C) to study their interactions with AuNPs functionalized with anionic ligands citrate, 3-mercaptopropionic acid, glutathione, and cationic ligand cysteamine. We studied the interactions by steady-state and time-resolved spectroscopic studies using membrane-sensitive probes 6-propionyl-2-dimethylaminonaphthalene (PRODAN) and 8-anilino-1 naphthalenesulfonate (ANS), as well as by confocal laser scanning microscopy (CLSM) imaging and dynamic light scattering (DLS) measurements. We observe that AuNPs bring in stability to the lipid vesicle, and the extent of interaction differs with the different surface ligands on the AuNPs. We observe that AuNPs functionalized with citrate effectively increase the phase-transition temperature of the vesicles by interacting with them. Our study reveals that the extent of interaction depends on the bulkiness of the ligands attached to the AuNPs. The bulkier ligands exert less van der Waals force, resulting in a weaker interaction. Moreover, we find that the interactions are more strongly pronounced when the vesicles are near the phase-transition temperature of the lipid.  The CLSM imaging and DLS measurements demonstrate the surface modifications in the vesicles as a result of these interactions.

Spectroscopic evidence for hydration and dehydration of lipid bilayers upon interaction with metal ions: a new physical insight.

In this manuscript, we investigate the interactions of different metal ions with zwitterionic phospholipid bilayers of different chain lengths using the well-known membrane probe PRODAN and steady state and time resolved fluorescence spectroscopy. We used three zwitterionic lipids that are widely different in their phase transition temperature, namely, dipalmitoylphosphatidylcholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (POPC) and salts of zinc (Zn), calcium (Ca) and magnesium (Mg). The steady state and time resolved studies reveal that the affinity of the metal ions follows the order Zn2+ > Ca2+ > Mg2+. The study further reveals that the lipid membrane with an unsaturated chain exhibits very small affinity towards metal ions. We find that the Zn2+ and Ca2+ metal ions induce significant gelation in the lipid bilayer possibly by dehydrating the lipid bilayer surface. The study also demonstrates that unlike Zn2+ and Ca2+, dehydration does not take place for Mg2+. The extreme hydration induced by Mg2+ is rationalized by the tight hydration of Mg2+ and very high free energy barrier of Mg2+ to bind with lipid oxygen as compared to that of water molecules.

TCR scanning of peptide/MHC through complementary matching of receptor and ligand molecular flexibility.

Although conformational changes in TCRs and peptide Ags presented by MHC protein (pMHC) molecules often occur upon binding, their relationship to intrinsic flexibility and role in ligand selectivity are poorly understood. In this study, we used nuclear magnetic resonance to study TCR-pMHC binding, examining recognition of the QL9/H-2L(d) complex by the 2C TCR. Although the majority of the CDR loops of the 2C TCR rigidify upon binding, the CDR3ß loop remains mobile within the TCR-pMHC interface. Remarkably, the region of the QL9 peptide that interfaces with CDR3ß is also mobile in the free pMHC and in the TCR-pMHC complex. Determination of conformational exchange kinetics revealed that the motions of CDR3ß and QL9 are closely matched. The matching of conformational exchange in the free proteins and its persistence in the complex enhances the thermodynamic and kinetic stability of the TCR-pMHC complex and provides a mechanism for facile binding. We thus propose that matching of structural fluctuations is a component of how TCRs scan among potential ligands for those that can bind with sufficient stability to enable T cell signaling.

Structural characterization of the C3 domain of cardiac myosin binding protein C and its hypertrophic cardiomyopathy-related R502W mutant.

Human cardiac myosin binding protein C (cMyBP-C), a thick filament protein found within the sarcomere of cardiac muscle, regulates muscle contraction and is essential for proper muscle function. Hypertrophic cardiomyopathy (HCM), a genetic disease affecting 1 in 500 people, is the major cause of death in young athletes. It is caused by genetic mutations within sarcomeric proteins. Forty-two percent of the HCM-related mutations are found in cMyBP-C. Here we present the nuclear magnetic resonance-derived structural ensembles of the wild-type cMyBP-C C3 domain and its HCM-related R502W mutant. The C3 domain adopts an immunoglobulin-like fold, and mutation of the exposed Arg502 to a tryptophan does not perturb its structure, dynamics, or stability. However, the R502W mutation does alter the predicted electrostatic properties of the C3 domain. We hypothesize that this mutation, and other HCM-linked mutations found within the same domain, may directly disrupt the interaction of cMyBP-C with other sarcomeric proteins.

Experimental methods to study the structure and dynamics of intrinsically disordered regions in proteins.

Eukaryotic proteins often feature long stretches of amino acids that lack a well-defined three-dimensional structure and are referred to as intrinsically disordered proteins (IDPs) or regions (IDRs). Although these proteins challenge conventional structure-function paradigms, they play vital roles in cellular processes. Recent progress in experimental techniques, such as NMR spectroscopy, single molecule FRET, high speed AFM and SAXS, have provided valuable insights into the biophysical basis of IDP function. This review discusses the advancements made in these techniques particularly for the study of disordered regions in proteins. In NMR spectroscopy new strategies such as 13C detection, non-uniform sampling, segmental isotope labeling, and rapid data acquisition methods address the challenges posed by spectral overcrowding and low stability of IDPs. The importance of various NMR parameters, including chemical shifts, hydrogen exchange rates, and relaxation measurements, to reveal transient secondary structures within IDRs and IDPs are presented. Given the high flexibility of IDPs, the review outlines NMR methods for assessing their dynamics at both fast (ps-ns) and slow (µs-ms) timescales. IDPs exert their functions through interactions with other molecules such as proteins, DNA, or RNA. NMR-based titration experiments yield insights into the thermodynamics and kinetics of these interactions. Detailed study of IDPs requires multiple experimental techniques, and thus, several methods are described for studying disordered proteins, highlighting their respective advantages and limitations. The potential for integrating these complementary techniques, each offering unique perspectives, is explored to achieve a comprehensive understanding of IDPs.

Stability and dynamics of extradenticle modulates its function.

Extradenticle (EXD) is a partner protein of the HOX transcription factors and plays an important role in the development of Drosophila. It confers increased affinity and specificity of DNA-binding to the HOX proteins. However, the DNA-binding homeodomain of EXD has a significantly weaker affinity to DNA compared to the HOX homeodomains. Here, we show that a glycine residue (G290) in the middle of the EXD DNA-binding helix primarily results in this weaker binding. Glycine destabilizes helices. To probe its role in the stability and function of the protein, G290 was mutated to alanine. The intrinsic stability of the DNA-binding helix increased in the G290A mutant as observed by NMR studies and molecular dynamics (MD) simulation. Also, NMR dynamics and MD simulation show that dynamic motions present in the wild-type protein are quenched in the mutant. This in turn resulted in increased stability of the entire homeodomain (ΔΔGG→A of -2.6 kcal/mol). Increased protein stability resulted in three-fold better DNA-binding affinity of the mutant as compared to the wild-type protein. Molecular mechanics with generalized Born and surface area solvation (MMGBSA) analysis of our MD simulation on DNA-bound models of both wild-type and mutant proteins shows that the contribution to binding is enhanced for most of the interface residues in the mutant compared to the wild-type. Interestingly, the flexible N-terminal arm makes more stable contact with the DNA minor groove in the mutant. We found that the two interaction sites i.e. the DNA-binding helix and the unstructured N-terminal arm influence each other via the bound DNA. These results provide an interesting conundrum: alanine at position 290 enhances both the stability and the DNA-binding affinity of the protein, however, evolution prefers glycine at this position. We have provided several plausible explanations for this apparent conundrum. The function of the EXD as a HOX co-factor requires its ability to discriminate similar DNA sequences, which is most likely comprom.

The intrinsically disordered transactivation region of HOXA9 regulates its function by auto-inhibition of its DNA-binding activity.

HOXA9 transcription factor is expressed in hematopoietic stem cells and is involved in the regulation of their differentiation and maturation to various blood cells. HOXA9 is linked to various leukemia and is a marker for poor prognosis of acute myeloid leukemia (AML). This protein has a conserved DNA-binding homeodomain and a transactivation domain. We show that this N-terminal transactivation domain is intrinsically disordered and inhibits DNA-binding by the homeodomain. Using NMR spectroscopy and molecular dynamics simulation, we show that the hexapeptide 197AANWLH202 in the disordered region transiently occludes the DNA-binding interface. The hexapeptide also forms a rigid segment, as determined by NMR dynamics, in an otherwise flexible disordered region. Interestingly, this hexapeptide is known to mediate the interaction of HOXA9 and its TALE partner proteins, such as PBX1, and help in cooperative DNA binding. Mutation of tryptophan to alanine in the hexapeptide abrogates the DNA-binding auto-inhibition. We propose that the disordered transactivation region plays a dual role in the regulation of HOXA9 function. In the absence of TALE partners, it inhibits DNA binding, and in the presence of TALE partners it interacts with the TALE protein and facilitates the cooperative DNA binding by the HOX-TALE complex.

Non-enzymatic glycation of human angiogenin: Effects on enzymatic activity and binding to hRI and DNA.

The effects of non-enzymatic glycation on the structural and functional properties of human angiogenin (hAng) have been investigated with respect to the formation of advanced glycated end products (AGEs), on prolonged treatment with d-Glucose, d-Fructose and d-Ribose at 37 °C. Fluorescence studies show the formation of fluorescent AGEs which exhibit emission maxima at 406 nm and 435 nm. Glycation of hAng with ribose leads to the maximum loss of its functional characteristic properties, as compared to fructose and glucose, along with the formation of higher oligomers. An increase in the incubation time results in the formation of higher oligomers with a concomitant decrease in the ribonucleolytic activity. The increase in the hydrodynamic radii of the glycated samples compared to native hAng is indicative of structural perturbations. The ribonucleolytic activity and the DNA binding ability of glycated hAng has been investigated by an agarose gel-based assay. Glycated hAng was unable to bind with human placental ribonuclease inhibitor (hRI), otherwise known to form one of the strongest protein-protein interaction systems with an affinity in the femtomolar range.

Deciphering the conformational stability of MazE7 antitoxin in Mycobacterium tuberculosis from molecular dynamics simulation study.

MazEF Toxin-antitoxin (TA) systems are associated with the persistent phenotype of the pathogen, Mycobacterium tuberculosis (Mtb), aiding their survival. Though extensively studied, the mode of action between the antitoxin-toxin and DNA of this family remains largely unclear. Here, the important interactions between MazF7 toxin and MazE7 antitoxin, and how MazE7 binds its promoter/operator region have been studied. To elucidate this, molecular dynamics (MD) simulation has been performed on MazE7, MazF7, MazEF7, MazEF7-DNA, and MazE7-DNA complexes to investigate how MazF7 and DNA affect the conformational change and dynamics of MazE7 antitoxin. This study demonstrated that the MazE7 dimer is disordered and one monomer (Chain C) attains stability after binding to the MazF7 toxin. Both the monomers (Chain C and Chain D) however are stabilized when MazE7 binds to DNA. MazE7 is also observed to sterically inhibit tRNA from binding to MazF7, thus suppressing its toxic activity. Comparative structural analysis performed on all the available antitoxins/antitoxin-toxin-DNA structures revealed MazEF7-DNA mechanism was similar to another TA system, AtaRT_E.coli. Simulation performed on the crystal structures of AtaR, AtaT, AtaRT, AtaRT-DNA, and AtaR-DNA showed that the disordered AtaR antitoxin attains stability by AtaT and DNA binding similar to MazE7. Based on these analyses it can thus be hypothesized that the disordered antitoxins enable tighter toxin and DNA binding thus preventing accidental toxin activation. Overall, this study provides crucial structural and dynamic insights into the MazEF7 toxin-antitoxin system and should provide a basis for targeting this TA system in combating Mycobacterium tuberculosis.Communicated by Ramaswamy H. Sarma.

Thermodynamics of the Association of Aminoglycoside Antibiotics with Human Angiogenin.

BACKGROUND: The body needs to maintain a firm balance between the inducers and inhibitors of angiogenesis, the process of proliferation of blood vessels from pre-existing ones. Human angiogenin (hAng), being a potent inducer of angiogenesis, is a cause of tumor cell proliferation, therefore its inhibition becomes a vital area of research. Aminoglycosides are linked ring systems consisting of amino sugars and an aminocyclitol ring and are in use in clinical practices for a long time. These compounds have found clinical uses as antibacterial agents that inhibit bacterial protein synthesis. OBJECTIVE: Gentamycin C1, Kanamycin A, Neomycin B, Paromomycin I, and Streptomycin A are commonly used aminoglycoside antibiotics that have been used for the present study. Among these, Neomycin has reported inhibitory activity against angiogenin-induced angiogenesis on the chicken chorioallantoic membrane. This study focuses on the thermodynamic parameters involved in the interactions of these antibiotics with hAng. METHODS: Agarose gel-based assay, Fluorescence quenching studies and Docking studies. RESULTS: Anti-ribonucleolytic effect of the antibiotics was observed qualitatively using an agarose gelbased assay, which shows that Neomycin exhibits the most efficient inhibition of hAng. Fluorescence quenching studies at different temperatures, using Stern-Volmer and van't Hoff equations provide information about the thermodynamics of binding, which furthermore highlights the higher binding constant of Neomycin. Docking studies showed that the antibiotics preferably interact with the nuclear translocation site, except Streptomycin, which shows affinity towards the ribonucleolytic site of the protein with very less affinity value. CONCLUSION: The study has shown the highly spontaneous formation of Neomycin-hAng complex, giving an exothermic reaction with increase in the degree of freedom of the protein-ligand complex.

Extein residues regulate the catalytic function of Spl DnaX intein enzyme by restricting the near-attack conformations of the active-site residues.

Intein enzymes catalyze the splicing of their flanking polypeptide chains and have found tremendous biotechnological applications. Their terminal residues form the catalytic core and participate in the splicing reaction. Hence, the neighboring N- and C-terminal extein residues influence the catalytic rate. As these extein residues vary depending on the substrate identity, we tested the influence of 20 amino acids at these sites in the Spl DnaX intein and observed significant variation of spliced product as well as N- and C-terminus cleavage product formation. We investigated the dependence of these reactions on the extein residues by molecular dynamics (MD) simulations on eight extein variants, and found that the conformational sampling of the active-site residues of the intein enzyme differed among these extein variants. We found that the extein variants that sample higher population of near-attack conformers (NACs) of the active-site residues undergo higher product formation in our activity assays. Ground state conformers that closely resemble the transition state are referred to as NACs. Very good correlation was observed between the NAC populations from the MD simulations of eight extein variants and the corresponding product formation from our activity assays. Furthermore, this molecular detail enabled us to elucidate the mechanistic roles of several conserved active-site residues in the splicing reaction. Overall, this study shows that the catalytic power of Spl DnaX intein enzyme, and most likely other inteins, depends on the efficiency of formation of NACs in the ground state, which is further modulated by the extein residues.

Complete thermodynamic and kinetic characterization of the isomer-specific interaction between Pin1-WW domain and the amyloid precursor protein cytoplasmic tail phosphorylated at Thr668.

Peptidyl prolyl cis-trans isomerization acts as an effective molecular timer that plays significant roles in biological and pathological processes. Enzymes such as Pin1 catalyze cis-trans isomerization, accelerating the otherwise slow isomerization rate into time scales relevant for cellular signaling. Here we have combined NMR line shape analysis, fluorescence spectroscopy, and isothermal titration calorimetry to determine the kinetic and thermodynamic parameters describing the trans-specific interaction between the binding domain of Pin1 (WW domain) and a key cis-trans molecular switch in the amyloid precursor protein cytoplasmic tail. A three-state model, in which the cis-trans isomerization equilibrium is coupled to the binding equilibrium through the trans isomer, was found to fit the data well. The trans isomer binds the WW domain with ∼22 µM affinity via very fast association (approaching the diffusion limit) and dissociation rates. The common structural and electrostatic characteristics of Pin1 substrates, which contain a phosphorylated serine/threonine-proline motif, suggest that very rapid binding kinetics are a general feature of Pin1 interactions with other substrates. The fast binding kinetics of the WW domain allows rapid response of Pin1 to the dynamic events of phosphorylation and dephosphorylation in the cell that alter the relative populations of diverse Pin1 substrates. Furthermore, our results also highlight the vastly different rates at which slow uncatalyzed cis-trans isomerization and fast isomer-specific binding events occur. These results, along with the experimental methods presented herein, should guide future experiments aimed at the thermodynamic and kinetic characterization of cis-trans molecular switches and isomer-specific interactions involved in various biological processes.

Elucidation of a pH-folding switch in the Pseudomonas syringae effector protein AvrPto.

Pathogenic bacteria have developed extraordinary strategies for invading host cells. The highly conserved type III secretion system (T3SS) provides a regulated conduit between the bacterial and host cytoplasm for delivery of a specific set of bacterial effector proteins that serve to disrupt host signaling and metabolism for the benefit of the bacterium. Remarkably, the inner diameter of the T3SS apparatus requires that effector proteins pass through in at least a partially unfolded form. AvrPto, an effector protein of the plant pathogen Pseudomonas syringae, adopts a helical bundle fold of low stability (DeltaG(F-->U) = 2 kcal/mol at pH 7, 26.6 degrees C) and offers a model system for chaperone-independent secretion. P. syringae effector proteins encounter a pH gradient as they translocate from the bacterial cytoplasm (mildly acidic) into the host cell (neutral). Here, we demonstrate that AvrPto possesses a pH-sensitive folding switch controlled by conserved residue H87 that operates precisely in the pH range expected between the bacterial and host cytoplasm environments. These results provide a mechanism for how a bacterial effector protein employs an intrinsic pH sensor to unfold for translocation via the T3SS and refold once in the host cytoplasm and provide fundamental insights for developing strategies for delivery of engineered therapeutic proteins to target tissues.