Mapping the transition state for a binding reaction between ancient intrinsically disordered proteins

The folding pathway of ubiquitin from all-atom molecular dynamics simulations

The folding pathways of some proteins include the population of partially structured species en route to the native state. Identification and characterization of these folding intermediates are particularly difficult as they are often only transiently populated and play different mechanistic roles, being either on-pathway productive species or off-pathway kinetic traps. To define the role of folding intermediates, a quantitative analysis of the folding and unfolding rate constants over a wide range of denaturant concentration is often required. Such a task is further complicated by the reversible nature of the folding reaction, which implies the observed kinetics to be governed by a complex combination of different microscopic rate constants. Here, we tackled this problem by measuring directly the folding rate constant under highly denaturing conditions, namely by inducing the folding of a PDZ domain through a quasi-irreversible binding reaction with a specific peptide. In analogy with previous works based on hydrogen exchange experiments, we present evidence that the folding pathway of the PDZ domain involves the formation of an obligatory on-pathway intermediate. The results presented exemplify a novel type of kinetic test to detect an on-pathway folding intermediate.

The Impact of Gene Expression Regulation on Evolution of Extracellular Signaling Pathways

The small ubiquitin-like modifier (SUMO) is involved in various cellular processes and mediates known non-covalent protein-protein interactions by three distinct binding surfaces, whose interactions are termed class I to class III. While interactors for the class I interaction, which involves binding of a SUMO-interacting motif (SIM) to a hydrophobic groove in SUMO-1 and SUMO-2/3, are widely abundant, only a couple of examples have been reported for the other two types of interactions. Class II binding is conveyed by the E67 loop region on SUMO-1. Many previous studies to identify SUMO binders using pull-down or microarray approaches did not strategize on the SUMO binding mode. Identification of SUMO binding partners is further complicated due to the typically transient and low affinity interactions with the modifier. Here we aimed to identify SUMO-1 binders selectively enriched for class II binding. Using a genetically encoded photo-crosslinker approach, we have designed SUMO-1 probes to covalently capture class II SUMO-1 interactors by strategically positioning the photo-crosslinking moiety on the SUMO-1 surface. The probes were validated using known class II and class I binding partners. We utilized the probe with p-benzoyl-phenylalanine (BzF, also termed BpF or Bpa) at the position of Gln69 to identify binding proteins from mammalian cell extracts using mass spectrometry. By comparison with results obtained with a similarly designed SUMO-1 probe to target SIM-mediated binders of the class I type, we identified 192 and 96 proteins specifically enriched by either probe, respectively. The implicated preferential class I or class II binding modes of these proteins will further contribute to unveiling the complex interplay of SUMO-1-mediated interactions.

A simple method for determining KAs of both low and high affinity IgG antibodies

Peripheral membrane proteins (PMPs) are critical mediators of signaling cascades initiated via activation of cell-surface receptors. Their functions rely on their innate ability to interact with membranes dynamically in response to rapidly changing cellular conditions. This membrane recruitment may occur via high-affinity interactions with specific lipids or transient, low-affinity membrane interactions. These weak and dynamic interactions that are critical regulators of protein function are challenging to capture. Taking Bruton’s Tyrosine Kinase (BTK), a non-receptor tyrosine kinase essential for B cell activation, as an example, we demonstrate a native mass spectrometry (nMS) platform to understand the recruitment of PMPs by directly studying it from lipid bilayers customized to target membranes. Our data demonstrates that BTK recognizes phosphatidylserine (PS) through sites distinct from phosphatidylinositol (3,4,5) phosphate (PIP3) binding. We show PS-bound BTK retains PIP3 binding simultaneously via high-affinity sites, exhibiting PIP3-independent basal membrane recruitment of BTK. Biochemical assays show that this PS-mediated recruitment sensitizes BTK to PIP3-mediated activation under near-physiological concentrations of PIP3. Thus, we propose a two-step model for BTK membrane recruitment and activation. A low-affinity interaction with high-copy number PS enables plasma membrane recruitment of BTK and increases its membrane-bound concentration. Upon B-cell activation, this pre-recruited, membrane-bound BTK population localizes to PIP3-rich domains through electrostatic gliding along the membrane driven by low-affinity PS and high-affinity PIP3 binding. This indicates a cooperative mechanism where PS can amplify B-cell signaling through increased membrane-bound BTK concentration. Our work demonstrates a general model of PH-domain-containing protein regulation by weak protein-lipid interactions, which can be extended to many other PMPs.

The interactions of fibronectin (Fn) with group B streptococci (GBS) were investigated using the atomic force microscope (AFM) and surface plasmon resonance (SPR) biosensing. Submonolayer amounts of Fn were immobilized onto the AFM tip by two different methods, using either a sulfosuccinimidyl-4-(N-maleimidomethyl) cycholhexane-1-carboxylate (SMCC) linker or a pyridyldithio poly(ethylene glycol) succinimidylpropionate (NHS-PEG-PDP) linker. Each step of both immobilization methods was characterized using x-ray photoelectron spectroscopy. Time-of-flight secondary ion mass spectrometry experiments indicated both methods produced Fn immobilized in a similar conformation. AFM force-distance curves from live GBS plated onto polystyrene exhibited several types of interactions between the Fn functionalized AFM tip and the surface of capsule-deficient GBS (no interactions, interactions with the cell wall, Fn unfolding, large specific unbinding events, and small specific unbinding events). From analysis of the force-distance curves that exhibited only a single specific unbinding event, the work of adhesion and rupture force for the SMCC immobilized Fn tips (11,131 pN nm and 213 pN) were larger than the corresponding values for the NHS-PEG-PDP immobilized Fn tips (8115 pN nm and 189 pN). The unbinding event occurred at distances approximately 100 nm further from the surface with the NHS-PEG-PDP immobilized Fn tip compared to SMCC immobilized Fn tip. The SPR experiments of soluble Fn with adsorbed serine protease C5a peptidase (Scp), the surface protein on GBS that binds Fn, showed that both low (millimolar) and high binding (nanomolar) affinity interactions were present. However, the low binding affinity interactions dominated the adsorption process and, with increasing Fn solution concentration, the amount of Scp bound to Fn via the high binding affinity interaction decreased. These data confirm that Scp binds only to adsorbed Fn at the Fn concentrations typically present in blood plasma.

There is a lack of experimental structural information about folding intermediates of multidomain proteins. Tick carboxypeptidase inhibitor (TCI) is a small, disulfide-rich protein consisting of two domains that fold and unfold autonomously through the formation of two major intermediates, IIIa and IIIb. Each intermediate contains three native disulfide bonds in one domain and six free cysteines in the other domain. Here we have determined the NMR structures of these two intermediates trapped and isolated at acidic pH in which they are stable and compared their structures with that of the native protein analyzed under the same conditions. Both IIIa and IIIb were found to contain a folded region that corresponds to the N- and C-terminal domains of TCI, respectively, with structures very similar to the corresponding regions of the native protein. The remainder of the polypeptide chains of the intermediates was shown to be unfolded in a random coil conformation. Solvent exchange measurements further indicated that the two protein domains are not completely independent, but affect each other in terms of dynamics and stability, in agreement with reported inhibitory activity data. The derived results provide structural evidence for symmetric TCI folding and unfolding mechanisms that converge in IIIa and IIIb and reveal the structural basis that accounts for the strong and simultaneous accumulation of both intermediates. Altogether, this work has important implications for a better understanding of the folding mechanisms of multidomain, disulfide-rich proteins.

Most proteins need to avoid the complex topologies when folding into the native structures, but some proteins with nontrivial topologies have been found in nature. Here we used protein unfolding simulations under high temperature and all-atom Gō-model to investigate the folding mechanisms for two trefoil knot proteins. Results show that, the contacts in β-sheet are important to the formation of knot protein, and if these contacts disappeared, the knot protein would be easy to untie. In the Gō-model simulations, the folding processes of the two knot proteins are similar. The compact structures of the two knot proteins with the native contacts in β-sheet are formed in transition state, and the intermediate state has loose C-terminal. This model also reveals the detailed folding mechanisms for the two proteins.

On the role of cooperativity in funneled energy landscapes.

An allosteric binding site at the human serotonin transporter mediates the inhibition of escitalopram by R-citalopram: Kinetic binding studies with the ALI/VFL–SI/TT mutant

Extracellular protein-protein interactions are essential for both intercellular communication and cohesion within multicellular organisms. Approximately a fifth of human genes encode membrane-tethered or secreted proteins, but they are largely absent from recent large-scale protein interaction datasets, making current interaction networks biased and incomplete. This discrepancy is due to the unsuitability of popular high-throughput methods to detect extracellular interactions because of the biochemical intractability of membrane proteins and their interactions. For example, cell surface proteins contain insoluble hydrophobic transmembrane regions, and their extracellular interactions are often highly transient, having half-lives of less than a second. To detect transient extracellular interactions on a large scale, we developed AVEXIS (avidity-based extracellular interaction screen), a high-throughput assay that overcomes these technical issues and can detect very transient interactions (half-lives <or= 0.1 sec) with a low false-positive rate. We used it to systematically screen for receptor-ligand pairs within the zebrafish immunoglobulin superfamily and identified novel ligands for both well-known and orphan receptors. Genes encoding receptor-ligand pairs were often clustered phylogenetically and expressed in the same or adjacent tissues, immediately implying their involvement in similar biological processes. Using AVEXIS, we have determined the first systematic low-affinity extracellular protein interaction network, supported by independent biological data. This technique will now allow large-scale extracellular protein interaction mapping in a broad range of experimental contexts.

The PAZ domain plays a key role in gene silencing pathway. The PAZ domain binds with siRNAs to form the multimeric RNA-induced silencing complex (RISC). RISC identifies mRNAs homologous to the siRNAs and promotes their degradation. It was found that binding with siRNA significantly enhances apo-PAZ folding. However, the mechanism by which folding is coupled to binding is poorly understood. Thus, the coupling relationship between binding and folding is very important for understanding the function of gene silencing. We have performed molecular dynamics (MD) of both bound and apo-PAZ to study the coupling mechanism between binding and folding in the siRNA-PAZ complex. Room-temperature MD simulations suggest that both PAZ and siRNA become more rigid and stable upon siRNA binding. Kinetic analysis of high-temperature MD simulations shows that both bound and apo-PAZ unfold via a two-state process. The unfolding pathways are different between bound and apo-PAZ: the order of helix III and helices I & II unfolding is switched. Furthermore, transition probability was used to determine the transition state ensemble for both bound and apo-PAZ. It was found that the transition state of bound PAZ is more compact than that of apo-PAZ. The predicted Φ-values suggest that the Φ-values of helix III and sheets of β3-β7 for bound PAZ are more native-like than those of apo-PAZ upon the binding of siRNA. The results can help us to understand the mechanism of gene silencing.

Time-resolved Fourier transform infrared spectroscopy (FTIR) in combination with photo-induced release of (18)O-labeled caged nucleotide has been employed to address mechanistic issues of GTP hydrolysis by Ras protein. Infrared spectroscopy of Ras complexes with nitrophenylethyl (NPE)-[alpha-(18)O(2)]GTP, NPE-[beta-(18)O(4)]GTP, or NPE-[gamma-(18)O(3)]GTP upon photolysis or during hydrolysis afforded a substantially improved mode assignment of phosphoryl group absorptions. Photolysis spectra of hydroxyphenylacyl-GTP and hydroxyphenylacyl-GDP bound to Ras and several mutants, Ras(Gly(12))-Mn(2+), Ras(Pro(12)), Ras(Ala(12)), and Ras(Val(12)), were obtained and yielded valuable information about structures of GTP or GDP bound to Ras mutants. IR spectra revealed stronger binding of GDP beta-PO(3)(2-) moiety by Ras mutants with higher activity, suggesting that the transition state is largely GDP-like. Analysis of the photolysis and hydrolysis FTIR spectra of the [beta-nonbridge-(18)O(2), alphabeta-bridge-(18)O]GTP isotopomer allowed us to probe for positional isotope exchange. Such a reaction might signal the existence of metaphosphate as a discrete intermediate, a key species for a dissociative mechanism. No positional isotope exchange was observed. Overall, our results support a concerted mechanism, but the transition state seems to have a considerable amount of dissociative character. This work demonstrates that time-resolved FTIR is highly suitable for monitoring positional isotope exchange and advantageous in many aspects over previously used methods, such as (31)P NMR and mass spectrometry.

The Limited Role of Nonnative Contacts in the Folding Pathways of a Lattice Protein