Understanding and control of structures and rates involved in protein ligand binding are essential for drug design. Unfortunately, atomistic molecular dynamics (MD) simulations cannot directly sample ...the excessively long residence and rearrangement times of tightly binding complexes. Here we exploit the recently developed multi-ensemble Markov model framework to compute full protein-peptide kinetics of the oncoprotein fragment
Mdm2 and the nano-molar inhibitor peptide PMI. Using this system, we report, for the first time, direct estimates of kinetics beyond the seconds timescale using simulations of an all-atom MD model, with high accuracy and precision. These results only require explicit simulations on the sub-milliseconds timescale and are tested against existing mutagenesis data and our own experimental measurements of the dissociation and association rates. The full kinetic model reveals an overall downhill but rugged binding funnel with multiple pathways. The overall strong binding arises from a variety of conformations with different hydrophobic contact surfaces that interconvert on the milliseconds timescale.
The mechanisms by which intrinsically disordered proteins engage in rapid and highly selective binding is a subject of considerable interest and represents a central paradigm to nuclear pore complex ...(NPC) function, where nuclear transport receptors (NTRs) move through the NPC by binding disordered phenylalanine-glycine-rich nucleoporins (FG-Nups). Combining single-molecule fluorescence, molecular simulations, and nuclear magnetic resonance, we show that a rapidly fluctuating FG-Nup populates an ensemble of conformations that are prone to bind NTRs with near diffusion-limited on rates, as shown by stopped-flow kinetic measurements. This is achieved using multiple, minimalistic, low-affinity binding motifs that are in rapid exchange when engaging with the NTR, allowing the FG-Nup to maintain an unexpectedly high plasticity in its bound state. We propose that these exceptional physical characteristics enable a rapid and specific transport mechanism in the physiological context, a notion supported by single molecule in-cell assays on intact NPCs.
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•Integrative structural biology reveals the basis of rapid nuclear transport•Transient binding of disordered nucleoporins leaves their plasticity unaffected•Multiple minimalistic low-affinity binding motifs create a polyvalent complex•A highly reactive and dynamic surface permits an ultrafast binding mechanism
Intrinsically disordered nucleoporins (Nups) engage rapidly with nuclear transport receptors through many minimalistic, weakly binding motifs. These Nups form polyvalent complexes while retaining conformational plasticity thus ensuring both rapid and specific transport.
Although protein-folding studies began several decades ago, it is only recently that the tools to analyze protein folding at the single-molecule level have been developed. Advances in single-molecule ...fluorescence and force spectroscopy techniques allow investigation of the folding and dynamics of single protein molecules, both at equilibrium and as they fold and unfold. The experiments are far from simple, however, both in execution and in interpretation of the results. In this review, we discuss some of the highlights of the work so far and concentrate on cases where comparisons with the classical experiments can be made. We conclude that, although there have been relatively few startling insights from single-molecule studies, the rapid progress that has been made suggests that these experiments have significant potential to advance our understanding of protein folding. In particular, new techniques offer the possibility to explore regions of the energy landscape that are inaccessible to classical ensemble measurements and, perhaps, to observe rare events undetectable by other means.
Proteins that fold cotranslationally may do so in a restricted configurational space, due to the volume occupied by the ribosome. How does this environment, coupled with the close proximity of the ...ribosome, affect the folding pathway of a protein? Previous studies have shown that the cotranslational folding process for many proteins, including small, single domains, is directly affected by the ribosome. Here, we investigate the cotranslational folding of an all-β Ig domain, titin I27. Using an arrest peptide-based assay and structural studies by cryo-EM, we show that I27 folds in the mouth of the ribosome exit tunnel. Simulations that use a kinetic model for the force dependence of escape from arrest accurately predict the fraction of folded protein as a function of length. We used these simulations to probe the folding pathway on and off the ribosome. Our simulations—which also reproduce experiments on mutant forms of I27—show that I27 folds, while still sequestered in the mouth of the ribosome exit tunnel, by essentially the same pathway as free I27, with only subtle shifts of critical contacts from the C to the N terminus.
Molecular evolution has focused on the divergence of molecular functions, yet we know little about how structurally distinct protein folds emerge de novo. We characterized the evolutionary ...trajectories and selection forces underlying emergence of β-propeller proteins, a globular and symmetric fold group with diverse functions. The identification of short propeller-like motifs (<50 amino acids) in natural genomes indicated that they expanded via tandem duplications to form extant propellers. We phylogenetically reconstructed 47-residue ancestral motifs that form five-bladed lectin propellers via oligomeric assembly. We demonstrate a functional trajectory of tandem duplications of these motifs leading to monomeric lectins. Foldability, i.e., higher efficiency of folding, was the main parameter leading to improved functionality along the entire evolutionary trajectory. However, folding constraints changed along the trajectory: initially, conflicts between monomer folding and oligomer assembly dominated, whereas subsequently, upon tandem duplication, tradeoffs between monomer stability and foldability took precedence.
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•Inferred 47-aminoacid ancestral motifs fold into functional β-propeller assemblies•Motif duplication, fusion, and diversification yield functional monomeric propellers•Folding efficiency was the key parameter optimized throughout propeller emergence•Single-motif precursors in extant genomes support the reconstructed emergence pathway
Experimental reconstruction of the emergence of a de novo protein indicates that “foldability” is the primary factor required for improved functionality along the entire evolutionary trajectory, although the parameters dictating optimal folding shifted as protein complexity increased.
The kinase-inducible domain interacting (KIX) domain of CREB binding protein binds to multiple intrinsically disordered transcription factors in vivo at two distinct sites on its surface. Several ...reports have been made of allosteric communication between these two sites in this well-characterized model system. In this work, we have performed fluorescence stopped-flow measurements to investigate the kinetics of binding of five KIX binding proteins. We find that they all have similar association and dissociation rate constants for complex formation, despite their wide range of intrinsic helical propensities. Furthermore, by careful arrangement of pseudofirst-order conditions, we have been able to show that both association and dissociation rate constants are decreased when a partner is bound at the alternative site. These decreases suggest that positive allosteric effects are not mediated by structural changes in binding sites but rather, through a more general mechanism, largely mediated through dissociation, which we propose is largely related to changes in the flexibility of the KIX domain itself.
Significance Specific protein–protein interactions are abundant in, and essential for, cellular life. In contrast to the well-studied docking of two already folded proteins, it has been recently ...established that many proteins are disordered and unfolded in the absence of their partner protein, but appear folded once bound. Must these initially disordered proteins transiently fold in isolation before binding their partners? We examine a small disordered protein and find that interactions with its (already structured) partner protein are what cause the relatively unstructured protein to fold. Thus, the requirement for one protein to fold is not an obstacle for reliable, fast association between two proteins. This result offers some explanation for the abundance of similar protein–protein interactions throughout biology.
Protein–protein interactions are at the heart of regulatory and signaling processes in the cell. In many interactions, one or both proteins are disordered before association. However, this disorder in the unbound state does not prevent many of these proteins folding to a well-defined, ordered structure in the bound state. Here we examine a typical system, where a small disordered protein (PUMA, p53 upregulated modulator of apoptosis) folds to an α-helix when bound to a groove on the surface of a folded protein (MCL-1, induced myeloid leukemia cell differentiation protein). We follow the association of these proteins using rapid-mixing stopped flow, and examine how the kinetic behavior is perturbed by denaturant and carefully chosen mutations. We demonstrate the utility of methods developed for the study of monomeric protein folding, including β-Tanford values, Leffler α, Φ-value analysis, and coarse-grained simulations, and propose a self-consistent mechanism for binding. Folding of the disordered protein before binding does not appear to be required and few, if any, specific interactions are required to commit to association. The majority of PUMA folding occurs after the transition state, in the presence of MCL-1. We also examine the role of the side chains of folded MCL-1 that make up the binding groove and find that many favor equilibrium binding but, surprisingly, inhibit the association process.
Association rates for interactions between folded proteins have been investigated extensively, allowing the development of computational and theoretical prediction methods. Less is known about ...association rates for complexes where one or more partner is initially disordered, despite much speculation about how they may compare to those for folded proteins. We have attached a fluorophore to the N-terminus of the 25 amino acid cMyb peptide used previously in NMR and equilibrium studies (termed FITC-cMyb), and used this to monitor the kinetics of its interaction with the KIX protein. We have investigated the ionic strength and temperature dependence of the kinetics, and conclude that the association process is extremely fast, apparently exceeding the rates predicted by formulations applicable to interactions between pairs of folded proteins. This is despite the fact that not all collisions result in complex formation (there is an observable activation energy for the association process). We propose that this is partially a result of the disordered nature of the FITC-cMyb peptide itself.
How do the key features of protein folding, elucidated from studies on native, isolated proteins, manifest in cotranslational folding on the ribosome? Using a well-characterized family of homologous ...α-helical proteins with a range of biophysical properties, we show that spectrin domains can fold vectorially on the ribosome and may do so via a pathway different from that of the isolated domain. We use cryo-EM to reveal a folded or partially folded structure, formed in the vestibule of the ribosome. Our results reveal that it is not possible to predict which domains will fold within the ribosome on the basis of the folding behavior of isolated domains; instead, we propose that a complex balance of the rate of folding, the rate of translation and the lifetime of folded or partly folded states will determine whether folding occurs cotranslationally on actively translating ribosomes.
Neighbouring domains of multidomain proteins with homologous tandem repeats have divergent sequences, probably as a result of evolutionary pressure to avoid misfolding and aggregation, particularly ...at the high cellular protein concentrations. Here we combine microfluidic-mixing single-molecule kinetics, ensemble experiments and molecular simulations to investigate how misfolding between the immunoglobulin-like domains of titin is prevented. Surprisingly, we find that during refolding of tandem repeats, independent of sequence identity, more than half of all molecules transiently form a wide range of misfolded conformations. Simulations suggest that a large fraction of these misfolds resemble an intramolecular amyloid-like state reported in computational studies. However, for naturally occurring neighbours with low sequence identity, these transient misfolds disappear much more rapidly than for identical neighbours. We thus propose that evolutionary sequence divergence between domains is required to suppress the population of long-lived, potentially harmful misfolded states, whereas large populations of transient misfolded states appear to be tolerated.
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