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In eukaryotes, many DNA/RNA-binding proteins possess intrinsically disordered regions (IDRs) with large negative charge, some of which involve a consecutive sequence of aspartate (D) or glutamate (E) residues. We refer to them as D/E repeats. The functional role of D/E repeats is not well understood, though some of them are known to cause autoinhibition through intramolecular electrostatic interaction with functional domains. In this work, we investigated the impacts of D/E repeats on the target DNA search kinetics for the high-mobility group box 1 (HMGB1) protein and the artificial protein constructs of the Antp homeodomain fused with D/E repeats of varied lengths. Our experimental data showed that D/E repeats of particular lengths can accelerate the target association in the overwhelming presence of non-functional high-affinity ligands (‘decoys’). Our coarse-grained molecular dynamics (CGMD) simulations showed that the autoinhibited proteins can bind to DNA and transition into the uninhibited complex with DNA through an electrostatically driven induced-fit process. In conjunction with the CGMD simulations, our kinetic model can explain how D/E repeats can accelerate the target association process in the presence of decoys. This study illuminates an unprecedented role of the negatively charged IDRs in the target search process.

Protein diffusion in lower-dimensional spaces is used for various cellular functions. For example, sliding on DNA is essential for proteins searching for their target sites, and protein diffusion on microtubules is important for proper cell division and neuronal development. On the one hand, these linear diffusion processes are mediated by long-range electrostatic interactions between positively charged proteins and negatively charged biopolymers and have similar characteristic diffusion coefficients. On the other hand, DNA and microtubules have different structural properties. Here, using computational approaches, we studied the mechanism of protein diffusion along DNA and microtubules by exploring the diffusion of both protein types on both biopolymers. We found that DNA-binding and microtubule-binding proteins can diffuse on each other's substrates; however, the adopted diffusion mechanism depends on the molecular properties of the diffusing proteins and the biopolymers. On the protein side, only DNA-binding proteins can perform rotation-coupled diffusion along DNA, with this being due to their higher net charge and its spatial organization at the DNA recognition helix. By contrast, the lower net charge on microtubule-binding proteins enables them to diffuse more quickly than DNA-binding proteins on both biopolymers. On the biopolymer side, microtubules possess intrinsically disordered, negatively charged C-terminal tails that interact with microtubule-binding proteins, thus supporting their diffusion. Thus, although both DNA-binding and microtubule-binding proteins can diffuse on the negatively charged biopolymers, the unique molecular features of the biopolymers and of their natural substrates are essential for function.

Replication protein A (RPA) plays a critical role in all eukaryotic DNA processing involving single-stranded DNA (ssDNA). Contrary to the notion that RPA provides solely inert protection to transiently formed ssDNA, the RPA–ssDNA complex acts as a dynamic DNA processing unit. Here, we studied the diffusion of RPA along 60 nt ssDNA using a coarse-grained model in which the ssDNA–RPA interface was modeled by both aromatic and electrostatic interactions. Our study provides direct evidence of bulge formation during the diffusion of ssDNA along RPA. Bulges can form at a few sites along the interface and store 1–7 nt of ssDNA whose release, upon bulge dissolution, leads to propagation of ssDNA diffusion. These findings thus support the reptation mechanism, which involves bulge formation linked to the aromatic interactions, whose short range nature reduces cooperativity in ssDNA diffusion. Greater cooperativity and a larger diffusion coefficient for ssDNA diffusion along RPA are observed for RPA variants with weaker aromatic interactions and for interfaces homogenously stabilized by electrostatic interactions. ssDNA propagation in the latter instance is characterized by lower probabilities of bulge formation; thus, it may fit the sliding-without-bulge model better than the reptation model. Thus, the reptation mechanism allows ssDNA mobility despite the extensive and high affinity interface of RPA with ssDNA. The short-range aromatic interactions support bulge formation while the long-range electrostatic interactions support the release of the stored excess ssDNA in the bulge and thus the overall diffusion.

Highly negatively charged segments containing only aspartate or glutamate residues (“D/E repeats”) are found in many eukaryotic proteins. For example, the C-terminal 30 residues of the HMGB1 protein are entirely D/E repeats. Using nuclear magnetic resonance (NMR), fluorescence, and computational approaches, we investigated how the D/E repeats causes the autoinhibition of HMGB1 against its specific binding to cisplatin-modified DNA. By varying ionic strength in a wide range (40–900 mM), we were able to shift the conformational equilibrium between the autoinhibited and uninhibited states toward either of them to the full extent. This allowed us to determine the macroscopic and microscopic equilibrium constants for the HMGB1 autoinhibition at various ionic strengths. At a macroscopic level, a model involving the autoinhibited and uninhibited states can explain the salt concentration-dependent binding affinity data. Our data at a microscopic level show that the D/E repeats and other parts of HMGB1 undergo electrostatic fuzzy interactions, each of which is weaker than expected from the macroscopic autoinhibitory effect. This discrepancy suggests that the multivalent nature of the fuzzy interactions enables strong autoinhibition at a macroscopic level despite the relatively weak intramolecular interaction at each site. Both experimental and computational data suggest that the D/E repeats interact preferentially with other intrinsically disordered regions (IDRs) of HMGB1. We also found that mutations mimicking post-translational modifications relevant to nuclear export of HMGB1 can moderately modulate DNA-binding affinity, possibly by impacting the autoinhibition. This study illuminates a functional role of the fuzzy interactions of D/E repeats.

DNA-binding proteins rely on linear diffusion along the longitudinal DNA axis, supported by their nonspecific electrostatic affinity for DNA, to search for their target recognition sites. One may therefore expect that the ability to engage in linear diffusion along DNA is universal to all DNA-binding proteins, with the detailed biophysical characteristics of that diffusion differing between proteins depending on their structures and functions. One key question is whether the linear diffusion mechanism is defined by translation coupled with rotation, a mechanism that is often termed sliding. We conduct coarse-grained and atomistic molecular dynamics simulations to investigate the minimal requirements for protein sliding along DNA. We show that coupling, while widespread, is not universal. DNA-binding proteins that slide along DNA transition to uncoupled translation-rotation (i.e., hopping) at higher salt concentrations. Furthermore, and consistently with experimental reports, we find that the sliding mechanism is the less dominant mechanism for some DNA-binding proteins, even at low salt concentrations. In particular, the toroidal PCNA protein is shown to follow the hopping rather than the sliding mechanism.

Intrinsically disordered regions (IDRs) of proteins are often characterized by a high fraction of charged residues, but differ in their overall net charge and in the organization of the charged residues. The function-encoding information stored via IDR charge composition and organization remains elusive. Here, we aim to decipher the sequence-function relationship in IDRs by presenting a comprehensive bioinformatic analysis of the charge properties of IDRs in the human, mouse, and yeast proteomes. About 50% of the proteins comprise at least a single IDR, which is either positively or negatively charged. Highly negatively charged IDRs are longer and possess greater net charge per residue compared with highly positively charged IDRs. A striking difference between positively and negatively charged IDRs is the characteristics of the repeated units, specifically, of consecutive Lys or Arg residues (K/R repeats) and Asp or Glu (D/E repeats) residues. D/E repeats are found to be about five times longer than K/R repeats, with the longest found containing 49 residues. Long stretches of consecutive D and E are found to be more prevalent in nucleic acid-related proteins. They are less common in prokaryotes, and in eukaryotes their abundance increases with genome size. The functional role of D/E repeats and the profound differences between them and K/R repeats are discussed.

Microtubules (MTs) are essential components of the eukaryotic cytoskeleton that serve as “highways” for intracellular trafficking. In addition to the well-known active transport of cargo by motor proteins, many MT-binding proteins seem to adopt diffusional motility as a transportation mechanism. However, because of the limited spatial resolution of current experimental techniques, the detailed mechanism of protein diffusion has not been elucidated. In particular, the precise role of tubulin tails and tail modifications in the diffusion process is unclear. Here, using coarse-grained molecular dynamics simulations validated against atomistic simulations, we explore the molecular mechanism of protein diffusion along MTs. We found that electrostatic interactions play a central role in protein diffusion; the disordered tubulin tails enhance affinity but slow down diffusion, and diffusion occurs in discrete steps. While diffusion along wild-type MT is performed in steps of dimeric tubulin, the removal of the tails results in a step of monomeric tubulin. We found that the energy barrier for diffusion is larger when diffusion on MTs is mediated primarily by the MT tails rather than the MT body. In addition, globular proteins (EB1 and PRC1) diffuse more slowly than an intrinsically disordered protein (Tau) on MTs. Finally, we found that polyglutamylation and polyglycylation of tubulin tails lead to slower protein diffusion along MTs, although polyglycylation leads to faster diffusion across MT protofilaments. Taken together, our results explain experimentally observed data and shed light on the roles played by disordered tubulin tails and tail modifications in the molecular mechanism of protein diffusion along MTs.

Summary The respiratory and intestinal tracts are exposed to physical and biological hazards accompanying the intake of air and food. Likewise, the vasculature is threatened by inflammation and trauma. Mucin glycoproteins and the related von Willebrand factor guard the vulnerable cell layers in these diverse systems. Colon mucins additionally house and feed the gut microbiome. Here, we present an integrated structural analysis of the intestinal mucin MUC2. Our findings reveal the shared mechanism by which complex macromolecules responsible for blood clotting, mucociliary clearance, and the intestinal mucosal barrier form protective polymers and hydrogels. Specifically, cryo-electron microscopy and crystal structures show how disulfide-rich bridges and pH-tunable interfaces control successive assembly steps in the endoplasmic reticulum and Golgi apparatus. Remarkably, a densely O-glycosylated mucin domain performs an organizational role in MUC2. The mucin assembly mechanism and its adaptation for hemostasis provide the foundation for rational manipulation of barrier function and coagulation., Graphical Abstract, Highlights • Mucin glycoprotein supramolecular assembly at Golgi pH promotes polymer formation • O-glycosylated regions help orchestrate mucin assembly • CysD domains have a novel fold, calcium binding motifs, and a role in mucin assembly • Mucins and von Willebrand factor share a mechanism to form linear polymers, Javitt et al. describe molecularly how mucin glycoproteins and von Willebrand factor—complex macromolecules responsible for mucociliary clearance in the lung, the intestinal mucosal barrier, and blood clotting—use a shared mechanism to form polymers and hydrogels that protect the respiratory tract, intestinal tract, and vasculature.

Entropy–enthalpy compensation is observed in many reactions, particularly for polymeric biomolecules that often involve large changes in entropy and enthalpy. The imperfect cancelation of entropy and enthalpy dictates many biophysical characteristics, such as protein thermodynamic stability and the free energy barrier for protein folding. In this study, we examine how tethering a conjugate to a protein may affect the thermodynamic stability of the protein. We found that a conjugate mostly affects the unfolded state by eliminating formation of some residual interactions. Consequently, both the enthalpy and the entropy of the unfolded state are affected. We suggest that, because this effect is not localized, the gain in conformational flexibility (i.e., increased entropy) is larger than the loss of some residual interaction (i.e., increased enthalpy). Therefore, the unfolded state of the conjugated protein has a lower free energy than that of the free protein, resulting in thermodynamic destabilization.

Predicting the effect of a single point mutation on protein thermodynamic stability (ΔΔ G) is an ongoing challenge with high relevance for both fundamental and applicable aspects of protein science. Drawbacks that limit the predictive power of stability prediction tools include the lack of representations for the explicit energetic terms of the unfolded state. Using coarse-grained simulations and analytical modeling analysis, we found that a mutation that involves the breaking of long-range contacts may lead to an increase in the unfolded state entropy, which can lead to an overall destabilization of the protein. A bioinformatics analysis indicates that the effect of mutation on the unfolded state is greater for hydrophobic or charged (compared with polar) residues that participate in long-range contacts through a loop length longer than 18 amino acids and whose formation probabilities are relatively high.

Many mutations that cause familial hypercholesterolemia localize to ligand-binding domain 5 (LA5) of the low-density lipoprotein receptor, motivating investigation of the folding and misfolding of this small, disulfide-rich, calcium-binding domain. LA5 folding is known to involve non-native disulfide isomers, yet these folding intermediates have not been structurally characterized. To provide insight into these intermediates, we used nuclear magnetic resonance (NMR) to follow LA5 folding in real time. We demonstrate that misfolded or partially folded disulfide intermediates are indistinguishable from the unfolded state when focusing on the backbone NMR signals, which provide information on the formation of only the final, native state. However, 13C labeling of cysteine side chains differentiated transient intermediates from the unfolded and native states and reported on disulfide bond formation in real time. The cysteine pairings in a dominant intermediate were identified using 13C-edited three-dimensiona...

The chaperonin GroEL and its co-chaperonin GroES form both GroEL–GroES bullet-shaped and GroEL–GroES(2) football-shaped complexes. The residence time of protein substrates in the cavities of these complexes is about 10 and 1 s, respectively. There has been much controversy regarding which of these complexes is the main functional form. Here, we show using computational analysis that GroEL protein substrates have a bimodal distribution of folding times, which matches these residence times, thereby suggesting that both bullet-shaped and football-shaped complexes are functional. More generally, co-existing complexes with different stoichiometries are not mutually exclusive with respect to having a functional role and can complement each other.

Proteins are subject to various conflicting forces that trade-off against each other. For example, during folding, the protein achieves lower enthalpy at the cost of lower entropy. Similarly, the trade-off for increased stability may be decreased flexibility, which may abolish allosteric pathways. Accordingly, stability trades-off against function, which may also trade-off against folding kinetics and mechanism. Furthermore, attaining increased stability may reduce a protein's ability to adopt novel functions. Understanding the biophysics and function of proteins requires quantification of the driving forces involved in each of the trade-offs. Indeed, quantification of the linkages in the network of trade-offs is essential to obtaining a more complete understanding of protein structure and function.

Microtubules (MTs), an essential component of the eukaryotic cytoskeleton, are a lattice of polymerized tubulin dimers and are crucial for various cellular processes. The genetic and chemical diversity of tubulin and their disordered tails gives rise to a “tubulin code”. The functional role of tubulin post-translational modifications (PTMs), which contribute to the chemical diversity of the tubulin code, is gradually being unraveled. However, variation in the length and spatial organization of tubulin poly-modifications leads to an enormous combinatorial PTM space, which is difficult to study experimentally. Hence, the impact of the combinatorial tubulin PTM space on the biophysical properties of tubulin tails and their interactions with other proteins remains elusive. Here, we combine all-atom and coarse-grained molecular dynamics simulations to elucidate the biophysical implications of the large combinatorial tubulin PTM space in the context of an MT lattice. We find that tail–body interactions are more dominant in the tubulin dimer than in an MT lattice, and are more significant for the tails of α compared with β tubulin. In addition, polyglutamylation, but not polyglycylation, expands the dimensions of the tubulin tails. Polyglutamylation also leads to a decrease in the diffusion rate of MT-associated protein EB1 on MTs, while polyglycylation often increases diffusion rate. These observations are generally not sensitive to the organization of the polymodifications. The effect of PTMs on MT charge density and tail dynamics are also discussed. Overall, this study presents a molecular quantification of the biophysical properties of tubulin tails and their polymodifications, and provides predictions on the functional importance of tubulin PTMs.