Structural characterization of intrinsically disordered proteins by NMR spectroscopy.

Recent advances in NMR methodology and techniques allow the structural investigation of biomolecules of increasing size with atomic resolution. NMR spectroscopy is especially well-suited for the study of intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs) which are in general highly flexible and do not have a well-defined secondary or tertiary structure under functional conditions. In the last decade, the important role of IDPs in many essential cellular processes has become more evident as the lack of a stable tertiary structure of many protagonists in signal transduction, transcription regulation and cell-cycle regulation has been discovered. The growing demand for structural data of IDPs required the development and adaption of methods such as 13C-direct detected experiments, paramagnetic relaxation enhancements (PREs) or residual dipolar couplings (RDCs) for the study of 'unstructured' molecules in vitro and in-cell. The information obtained by NMR can be processed with novel computational tools to generate conformational ensembles that visualize the conformations IDPs sample under functional conditions. Here, we address NMR experiments and strategies that enable the generation of detailed structural models of IDPs

Disordered proteins and network disorder in network descriptions of protein structure, dynamics and function. Hypotheses and a comprehensive review

During the last decade, network approaches became a powerful tool to describe protein structure and dynamics. Here we review the links between disordered proteins and the associated networks, and describe the consequences of local, mesoscopic and global network disorder on changes in protein structure and dynamics. We introduce a new classification of protein networks into ‘cumulus-type’, i.e., those similar to puffy (white) clouds, and ‘stratus-type’, i.e., those similar to flat, dense (dark) low-lying clouds, and relate these network types to protein disorder dynamics and to differences in energy transmission processes. In the first class, there is limited overlap between the modules, which implies higher rigidity of the individual units; there the conformational changes can be described by an ‘energy transfer’ mechanism. In the second class, the topology presents a compact structure with significant overlap between the modules; there the conformational changes can be described by ‘multi-trajectories’; that is, multiple highly populated pathways. We further propose that disordered protein regions evolved to help other protein segments reach ‘rarely visited’ but functionally-related states. We also show the role of disorder in ‘spatial games’ of amino acids; highlight the effects of intrinsically disordered proteins (IDPs) on cellular networks and list some possible studies linking protein disorder and protein structure networks

The Ensemble of Conformations of Antifreeze Glycoproteins (AFGP8): A Study Using Nuclear Magnetic Resonance Spectroscopy.

The primary sequence of antifreeze glycoproteins (AFGPs) is highly degenerate, consisting of multiple repeats of the same tripeptide, Ala-Ala-Thr*, in which Thr* is a glycosylated threonine with the disaccharide beta-d-galactosyl-(1,3)-alpha-N-acetyl-d-galactosamine. AFGPs seem to function as intrinsically disordered proteins, presenting challenges in determining their native structure. In this work, a different approach was used to elucidate the three-dimensional structure of AFGP8 from the Arctic cod Boreogadus saida and the Antarctic notothenioid Trematomus borchgrevinki. Dimethyl sulfoxide (DMSO), a non-native solvent, was used to make AFGP8 less dynamic in solution. Interestingly, DMSO induced a non-native structure, which could be determined via nuclear magnetic resonance (NMR) spectroscopy. The overall three-dimensional structures of the two AFGP8s from two different natural sources were different from a random coil ensemble, but their "compactness" was very similar, as deduced from NMR measurements. In addition to their similar compactness, the conserved motifs, Ala-Thr*-Pro-Ala and Ala-Thr*-Ala-Ala, present in both AFGP8s, seemed to have very similar three-dimensional structures, leading to a refined definition of local structural motifs. These local structural motifs allowed AFGPs to be considered functioning as effectors, making a transition from disordered to ordered upon binding to the ice surface. In addition, AFGPs could act as dynamic linkers, whereby a short segment folds into a structural motif, while the rest of the AFGPs could still be disordered, thus simultaneously interacting with bulk water molecules and the ice surface, preventing ice crystal growth

Buried and accessible surface area control intrinsic protein flexibility

Proteins experience a wide variety of conformational dynamics that can be crucial for facilitating their diverse functions. How is the intrinsic flexibility required for these motions encoded in their three-dimensional structures? Here, the overall flexibility of a protein is demonstrated to be tightly coupled to the total amount of surface area buried within its fold. A simple proxy for this, the relative solvent accessible surface area (Arel), therefore shows excellent agreement with independent measures of global protein flexibility derived from various experimental and computational methods. Application of Arel on a large scale demonstrates its utility by revealing unique sequence and structural properties associated with intrinsic flexibility. In particular, flexibility as measured by Arel shows little correspondence with intrinsic disorder, but instead tends to be associated with multiple domains and increased {\alpha}- helical structure. Furthermore, the apparent flexibility of monomeric proteins is found to be useful for identifying quaternary structure errors in published crystal structures. There is also a strong tendency for the crystal structures of more flexible proteins to be solved to lower resolutions. Finally, local solvent accessibility is shown to be a primary determinant of local residue flexibility. Overall this work provides both fundamental mechanistic insight into the origin of protein flexibility and a simple, practical method for predicting flexibility from protein structures.Comment: 36 pages, 11 figures, author's manuscript, accepted for publication in Journal of Molecular Biolog

Computational approaches for inferring the functions of intrinsically disordered proteins.

Intrinsically disordered proteins (IDPs) are ubiquitously involved in cellular processes and often implicated in human pathological conditions. The critical biological roles of these proteins, despite not adopting a well-defined fold, encouraged structural biologists to revisit their views on the protein structure-function paradigm. Unfortunately, investigating the characteristics and describing the structural behavior of IDPs is far from trivial, and inferring the function(s) of a disordered protein region remains a major challenge. Computational methods have proven particularly relevant for studying IDPs: on the sequence level their dependence on distinct characteristics determined by the local amino acid context makes sequence-based prediction algorithms viable and reliable tools for large scale analyses, while on the structure level the in silico integration of fundamentally different experimental data types is essential to describe the behavior of a flexible protein chain. Here, we offer an overview of the latest developments and computational techniques that aim to uncover how protein function is connected to intrinsic disorder

Targeting Intrinsically Disordered Proteins through Dynamic Interactions

Intrinsically disordered proteins (IDPs) are over-represented in major disease pathways and have attracted significant interest in understanding if and how they may be targeted using small molecules for therapeutic purposes. While most existing studies have focused on extending the traditional structure-centric drug design strategies and emphasized exploring pre-existing structure features of IDPs for specific binding, several examples have also emerged to suggest that small molecules could achieve specificity in binding IDPs and affect their function through dynamic and transient interactions. These dynamic interactions can modulate the disordered conformational ensemble and often lead to modest compaction to shield functionally important interaction sites. Much work remains to be done on further elucidation of the molecular basis of the dynamic small molecule–IDP interaction and determining how it can be exploited for targeting IDPs in practice. These efforts will rely critically on an integrated experimental and computational framework for disordered protein ensemble characterization. In particular, exciting advances have been made in recent years in enhanced sampling techniques, Graphic Processing Unit (GPU)-computing, and protein force field optimization, which have now allowed rigorous physics-based atomistic simulations to generate reliable structure ensembles for nontrivial IDPs of modest sizes. Such de novo atomistic simulations will play crucial roles in exploring the exciting opportunity of targeting IDPs through dynamic interactions

Toward a Molecular Mechanism of Phase Separation in Disordered Elastin-Like Proteins

Since the last decade, an increasing number of proteins have been shown to be capable of undergoing reversible liquid-liquid phase separation (LLPS) in response to an external stimulus, and the resulting protein-rich phase (coacervate) is considered as one of the main components of membrane-less organelles. Most of these proteins are intrinsically disordered proteins (IDPs) or contain intrinsically disordered regions. More importantly, LLPS often plays an important role in cellular signaling and development of cells and tissues. However, the molecular mechanisms underlying LLPS of proteins remain poorly understood. Elastin-like proteins (ELPs), a class of IDPs derived from the hydrophobic domains of tropoelastin, are known to undergo LLPS reversibly above a concentration-dependent transition temperature (TT), allowing ELPs to be a promising thermo-responsive drug delivery vector for treating cancer. Previous studies have suggested that, as temperature increases, ELPs experience an increased propensity for type II beta-turns. Our hypothesis is that the interaction is initiated at the beta-turn positions. In this work, integrative approaches including experimental and computational methods were employed to study the early stages of ELP phase separation. Using nuclear magnetic resonance spectroscopy (NMR), and paramagnetic relaxation enhancement (PRE), we have characterized structural properties of self-association in several ELPs. NMR chemical shifts suggest that ELPs adopt a beta-turn conformation even at temperatures below the TT. The intermolecular PRE reveals there is a stronger interaction between the higher beta-turn propensity regions. Building on this observation, a series of structural ensembles were generated for ELP incorporating differing amounts of beta-turn bias, from 1% to 90%. To mimic the early stages of the phase change, two monomers were paired, assuming preferential interaction at beta-turn regions. Following dimerization, the ensemble-averaged hydrodynamic properties were calculated for each degree of beta-turn bias, and results were compared with analytical ultracentrifugation (AUC) experiments at various temperatures. The ensemble calculation reveals that accessible surface area changes dramatically as oligomers are formed from monomers with a high beta-turn content. Together, these observations suggest a model where ELP self-association is initiated at beta-turn positions, where the driving force of phase separation is solvent exclusion due to changes in the hydrophobic accessible surface area

Multi-scale Simulations of Dynamic Protein Structures and Interactions

Intrinsically disordered proteins (IDPs) are functional proteins that lack stable tertiary structures in the unbound state. They frequently remain dynamic even within specific complexes and assemblies. IDPs are major components of cellular regulatory networks and have been associated with cancers, diabetes, neurodegenerative diseases, and other human diseases. Computer simulations are essential for deriving a molecular description of the disordered protein ensembles and dynamic interactions for mechanistic understanding of IDPs in biology, diseases, and therapeutics. However, accurate simulation of the heterogeneous ensembles and dynamic interactions of IDPs is extremely challenging because of both the prohibitive computational cost and demanding force field accuracy. In this dissertation, we developed a set of enhanced sampling and multi-scale simulation methods to overcome these limitations, and successfully applied them to study the structure, interaction and phase separation of IDPs. We have first applied the state-of-the-art explicit solvent atomistic simulations to study the inhibitory mechanism of the disordered N-terminal domain of Staphylococcal peroxidase inhibitor (SPIN). We performed high-temperature simulations to study the coupled binding and folding process during SPIN inhibition of the host myeloperoxidase (MPO) enzyme. The results showed that differences in dynamics may provide a physical basis of the ability of different SPIN homologs to inhibit innate immunity. Recognizing the need for enhanced sampling methods for IDP simulation, we have developed a new replica-exchange with solute tempering (REST) protocol to achieve more efficient explicit solvent sampling of disordered protein ensembles. We proposed that the scaling of protein-water interactions in REST is a free parameter that could be optimized to better control how the protein conformational properties (e.g., chain expansion) at different effective temperatures and achieve more effective sampling. Specifically, we developed a REST3 protocol that rebalances the protein-protein and protein-water interactions and eliminates the unanticipated chain collapse at high temperature conditions in the previous REST2 protocol. Application to model IDPs demonstrated that REST3 prevented the conformational segregation during exchanges, leading to an effective temperature random walk across all conditions and accelerating the simulation of the protein conformational space. Even with enhanced sampling, accurate description of disordered conformations at atomistic level remains extremely challenging for complex IDPs. Alternatively, coarse-grained simulations can provide an effective strategy for overcoming the length- and time-scale limitations. Here, we refined a hybrid-resolution coarse-grained model (HyRes) for accurate simulation of disordered protein ensembles and dynamic protein interactions. HyRes contains atomistic backbone and coarse-grained sidechain beads, to provide semi-quantitative description of residual secondary structures and long-range interactions. Specifically, we introduced a surface area-based implicit solvation energy term, and iteratively re-optimized the effective interaction strength potentials. The new model, referred as HyRes II, provides near quantitative descriptions of IDP long-range non-specific interactions and secondary structures, at a level comparable to the latest atomistic protein force fields. Applied to the disordered N-terminal transactivation domain (TAD) of tumor suppressor p53, HyRes II faithfully recapitulates various nontrivial structural properties to a level of accuracy that is comparable to a99SB-disp, one of the best atomistic protein force fields. Moreover, we demonstrate HyRes II’s efficacy in accurately simulating the dynamic interaction between TAD and the DNA-binding domain of p53, generating structural ensembles that align closely with existing NMR data. Encouraged by successes of HyRes II for probing dynamic interactions of IDPs, we further investigated its suitability for simulating IDP-mediated phase separation, which underlies the formation of biomolecular condensates and has attracted intensive interests. Compared to the popular single-bead models, HyRes has the potential to describe backbone-mediated interactions and capture the role of residual structures in phase separation. Reimplemented on GPU, our simulations showed that HyRes is efficient enough to directly simulate the spontaneous phase separation of IDPs and at the time balanced enough to capture the effects of mutational and structural perturbations. For peptide GY-23, HyRes simulations reveal increased ��-structures in condensates, which are consistent with experimental observations. For the conserved region (CR) of TDP-43, HyRes simulations successfully recapitulate the apparent correlation between helical propensities and condensate stability. In depth analysis, however, revealed that residual helices did not directly mediate interpeptide interactions to stabilize the condensed phase. Instead, it is the balance between backbone and sidechain-mediated interactions, as modulated by residual structures, that actually determines phase separation propensity. Finally, we have applied the HyRes II model to study the dynamic interaction of West Nile virus (WNV) NS2B/NS3 proteases with the ClyA protein nanopore. Nanopore tweezers provide a powerful approach for label-free detection of protein dynamics at the single-molecule level, by capturing the protein analyte in the lumen of the nanopore. From the steered-MD and standard MD simulations, we discovered that the protease could bind dynamically to a middle region of the ClyA nanopore, mediated mainly by electrostatically interactions. In particular, we identified a key Glu residue within the ClyA lumen, mutation of which to Ala or Lys could further stabilize the protease/nanopore interaction. This led to the design a modified ClyA nanopore tweezer that can stably capture the protease and resolve the dynamics between NS2B/NS3 open and closed conformations