Diffusing protein binders to intrinsically disordered proteins
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Design of intrinsically disordered protein variants with diverse structural properties
Intrinsically disordered proteins (IDPs) perform a broad range of functions in biology, suggesting that the ability to design IDPs could help expand the repertoire of proteins with novel functions. Computational design of IDPs with specific conformational properties has, however, been difficult because of their substantial dynamics and structural complexity. We describe a general algorithm for designing IDPs with specific structural properties. We demonstrate the power of the algorithm by generating variants of naturally occurring IDPs that differ in compaction, long-range contacts, and propensity to phase separate. We experimentally tested and validated our designs and analyzed the sequence features that determine conformations. We show how our results are captured by a machine learning model, enabling us to speed up the algorithm. Our work expands the toolbox for computational protein design and will facilitate the design of proteins whose functions exploit the many properties afforded by protein disorder.
Science & Technology - Other Topics↗
Toward understanding biomolecular materials comprising intrinsically disordered proteins via simulation and experiment
Different simulation approaches have been applied to understanding and predicting key features of the solution behavior of intrinsically disordered proteins.
Wang, Bin [Department of Materials Science and Eng↗
Structural Heterogeneity and Hydrodynamics of an Intrinsically Disordered Protein Condensate
Biology demonstrates precise control over the free-energy landscape through the selective partitioning of biomacromolecules into membraneless organelles, enabling essential functions such as biochemical transformations, signaling cascades, and mechanical reinforcement. Although the function of these condensates depends on their underlying structure and hydrodynamics, molecular-scale information on these systems remains sparse. Here, in this study, neutron scattering is used to probe the organization and dynamics of the intrinsically disordered N-terminal domain of Galectin-3, an extracellular lectin responsible for facilitating liquid–liquid phase separation on the cellular surface, in both dilute and condensed phases. Dilute solutions contain isolated protein chains in equilibrium with mesoscopic clusters, whereas the condensed phase adopts a bicontinuous, microemulsion-like morphology. The dilute phase behavior is quantitatively described by coarse-grained polymer models from soft-matter physics, demonstrating their predictive power for complex biological proteins. At elevated concentrations, the proteins self-assemble akin to block copolymers, microphase separating through the aggregation of hydrophobic domains along the protein contour. The resulting condensate remains fluid-like despite a 25-fold increase in concentration; its internal hydrodynamics slow by only a factor of 3 relative to dilute protein chains. These results provide a molecular-level framework for how disordered proteins achieve both the structural complexity and dynamic fluidity of biomolecular condensates.
Carrick, Brian R. [Massachusetts Inst. of Technolo↗
Conserved and repetitive motifs in an intrinsically disordered protein drive ⍺-carboxysome assembly
Not Available
Biochemistry & Molecular Biology↗
Functional in vitro diversity of an intrinsically disordered plant protein during freeze–thawing is encoded by its structural plasticity
Intrinsically disordered late embryogenesis abundant (LEA) proteins play a central role in the tolerance of plants and other organisms to dehydration brought upon, for example, by freezing temperatures, high salt concentration, drought or desiccation, and many LEA proteins have been found to stabilize dehydration-sensitive cellular structures. Their conformational ensembles are highly sensitive to the environment, allowing them to undergo conformational changes and adopt ordered secondary and quaternary structures and to participate in formation of membraneless organelles. In an interdisciplinary approach, we discovered how the functional diversity of the Arabidopsis thaliana LEA protein COR15A found in vitro is encoded in its structural repertoire, with the stabilization of membranes being achieved at the level of secondary structure and the stabilization of enzymes accomplished by the formation of oligomeric complexes. We provide molecular details on intra- and inter-monomeric helix–helix interactions, demonstrate how oligomerization is driven by an α-helical molecular recognition feature (α-MoRF) and provide a rationale that the formation of noncanonical, loosely packed, right-handed coiled-coils might be a recurring theme for homo- and hetero-oligomerization of LEA proteins.
59 BASIC BIOLOGICAL SCIENCES↗
Asymmetric oligomerization state and sequence patterning can tune multiphase condensate miscibility
Endogenous biomolecular condensates, composed of a multitude of proteins and RNAs, can organize into multiphasic structures with compositionally distinct phases. This multiphasic organization is generally understood to be critical for facilitating their proper biological function. However, the biophysical principles driving multiphase formation are not completely understood. Here we use in vivo condensate reconstitution experiments and coarse-grained molecular simulations to investigate how oligomerization and sequence interactions modulate multiphase organization in biomolecular condensates. We demonstrate that increasing the oligomerization state of an intrinsically disordered protein results in enhanced immiscibility and multiphase formation. Interestingly, we find that oligomerization tunes the miscibility of intrinsically disordered proteins in an asymmetric manner, with the effect being more pronounced when the intrinsically disordered protein, exhibiting stronger homotypic interactions, is oligomerized. Our findings suggest that oligomerization is a flexible biophysical mechanism that cells can exploit to tune the internal organization of biomolecular condensates and their associated biological functions.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
Ion-selective conformational stabilization of a disordered repeats-in-toxin protein domain
Ion-binding intrinsically disordered proteins (IDPs) recruit and bind to specific metal ions to perform critical biological functions. In proteins where ion binding and structural transitions are coupled, interactions with off-target toxic metals can dramatically disrupt protein structure and function, exemplified by lead and mercury poisoning. Understanding the complex mechanisms underlying how IDPs exclude or allow binding to different ionic species is crucial for addressing the origins of metal toxicity in biological systems. Here, we elucidate mechanisms of ion selectivity in an IDP that adopts a structure upon Ca 2+ binding. We probed ion-induced conformational changes of a repeats-in-toxin (RTX) protein domain in the presence of different ion ligands—Mg 2+ , Ca 2+ , Sr 2+ , and Ba 2+ —with chemical similarities but drastically different ionic radii. RTX adopts ion-selective conformations measured by x-ray crystallography, small-angle x-ray scattering, and circular dichroism. High-resolution x-ray structures reveal that Sr 2+ induces a nearly identical RTX structure as natively binding Ca 2+ , enabled by the intrinsic flexibility and disorder of the protein. Small-angle x-ray scattering and circular dichroism indicate that smaller Mg 2+ does not induce a significant conformational change in RTX, whereas larger Ba 2+ induces a partially folded structure. These results highlight the importance of geometric constraints imposed by protein structure in determining metal ion selectivity, yielding insights into how off-target ion binding may result in protein misfolding and malfunction.
Gudinas, Alana P. [Stanford Univ., CA (United Stat↗
How hydrophobicity, side chains, and salt affect the dimensions of disordered proteins
Abstract Despite the generally accepted role of the hydrophobic effect as the driving force for folding, many intrinsically disordered proteins (IDPs), including those with hydrophobic content typical of foldable proteins, behave nearly as self‐avoiding random walks (SARWs) under physiological conditions. Here, we tested how temperature and ionic conditions influence the dimensions of the N‐terminal domain of pertactin (PNt), an IDP with an amino acid composition typical of folded proteins. While PNt contracts somewhat with temperature, it nevertheless remains expanded over 10–58°C, with a Flory exponent, ν , >0.50. Both low and high ionic strength also produce contraction in PNt, but this contraction is mitigated by reducing charge segregation. With 46% glycine and low hydrophobicity, the reduced form of snow flea anti‐freeze protein (red‐sfAFP) is unaffected by temperature and ionic strength and persists as a near‐SARW, ν ~ 0.54, arguing that the thermal contraction of PNt is due to stronger interactions between hydrophobic side chains. Additionally, red‐sfAFP is a proxy for the polypeptide backbone, which has been thought to collapse in water. Increasing the glycine segregation in red‐sfAFP had minimal effect on ν . Water remained a good solvent even with 21 consecutive glycine residues ( ν > 0.5), and red‐sfAFP variants lacked stable backbone hydrogen bonds according to hydrogen exchange. Similarly, changing glycine segregation has little impact on ν in other glycine‐rich proteins. These findings underscore the generality that many disordered states can be expanded and unstructured, and that the hydrophobic effect alone is insufficient to drive significant chain collapse for typical protein sequences.
59 BASIC BIOLOGICAL SCIENCES↗
Structural studies of intrinsically disordered MLL -fusion protein AF9 in complex with peptidomimetic inhibitors
AF9 (MLLT3) and its paralog ENL(MLLT1) are members of the YEATS family of proteins with important role in transcriptional and epigenetic regulatory complexes. These proteins are two common MLL fusion partners in MLL -rearranged leukemias. The oncofusion proteins MLL-AF9/ENL recruit multiple binding partners, including the histone methyltransferase DOT1L, leading to aberrant transcriptional activation and enhancing the expression of a characteristic set of genes that drive leukemogenesis. The interaction between AF9 and DOT1L is mediated by an intrinsically disordered C-terminal ANC1 homology domain (AHD) in AF9, which undergoes folding upon binding of DOT1L and other partner proteins. We have recently reported peptidomimetics that disrupt the recruitment of DOT1L by AF9 and ENL, providing a proof-of-concept for targeting AHD and assessing its druggability. Intrinsically disordered proteins, such as AF9 AHD, are difficult to study and characterize experimentally on a structural level. In this study, we present a successful protein engineering strategy to facilitate structural investigation of the intrinsically disordered AF9 AHD domain in complex with peptidomimetic inhibitors by using maltose binding protein (MBP) as a crystallization chaperone connected with linkers of varying flexibility and length. The strategic incorporation of disulfide bonds provided diffraction-quality crystals of the two disulfide-bridged MBP–AF9 AHD fusion proteins in complex with the peptidomimetics. These successfully determined first series of 2.1–2.6 Å crystal complex structures provide high-resolution insights into the interactions between AHD and its inhibitors, shedding light on the role of AHD in recruiting various binding partner proteins. We show that the overall complex structures closely resemble the reported NMR structure of AF9 AHD/DOT1L with notable difference in the conformation of the β-hairpin region, stabilized through conserved hydrogen bonds network. These first series of AF9 AHD/peptidomimetics complex structures are providing insights of the protein–inhibitor interactions and will facilitate further development of novel inhibitors targeting the AF9/ENL AHD domain.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
Use of Tardigrade Proteins for Enhancing Cellular Stress Tolerance on the Moon
With sights set on returning a human presence to the Moon and plans to explore even farther beyond, it is imperative we understand the impact of deep space radiation, partial gravity, and the lunar surface on biology. The need for countermeasures to protect future astronauts from the unique conditions of space is ever-pressing and an assessment of bioproduction capabilities in space is a crucial step for sustaining long-term missions. The Lunar Explorer Instrument for space biology Applications (LEIA) aims to investigate the cell’s response to the lunar environment and test strategies for enhancing cellular tolerance on the Moon, utilizing the budding yeast Saccharomyces cerevisiae as a biosensor. S. cerevisiae serves as an excellent model organism due to its widespread use as an analog for human cells, well-characterized set of genetic tools, and ability to survive desiccation. However, these cells must remain desiccated for up to one year prior to launch and the radiation-sensitive strains designed for LEIA will likely be more vulnerable to desiccation effects. In this work, we introduce tardigrade proteins known for their protective mechanisms into potential LEIA strains to test genetic strategies for improving desiccation and radiation tolerance. Tardigrades are extremotolerant animals well-known for their ability to survive in harsh environments. Notably, they can survive extreme desiccation and radiation due to the upregulation of intrinsically disordered proteins (IDPs). It has been shown that integration of these tardigrade IDPs, such as cytosolic abundant heat-soluble (CAHS) proteins, into yeast can confer increased desiccation tolerance in synergy with trehalose. Our ongoing testing and assessment of bioengineered radiation and desiccation tolerance will help us gain insight into optimizing strain design and selection for protecting LEIA’s biological payload as well as genetic engineering strategies for enhancing bioproduction potential for future missions beyond low Earth orbit.
Genetics↗
Use of Tardigrade Proteins for Enhancing Cellular Stress Tolerance on the Moon
With sights set on returning a human presence to the Moon and plans to explore even farther beyond, it is imperative we understand the impact of deep space radiation, partial gravity, and the lunar surface on biology. The need for countermeasures to protect future astronauts from the unique conditions of space is ever-pressing and an assessment of bioproduction capabilities in space is a crucial step for sustaining long-term missions. The Lunar Explorer Instrument for space biology Applications (LEIA) aims to investigate the cell’s response to the lunar environment and test strategies for enhancing cellular tolerance on the Moon, utilizing the budding yeast Saccharomyces cerevisiae as a biosensor. S. cerevisiae serves as an excellent model organism due to its widespread use as an analog for human cells, well-characterized set of genetic tools, and ability to survive desiccation. However, these cells must remain desiccated for up to one year prior to launch and the radiation-sensitive strains designed for LEIA will likely be more vulnerable to desiccation effects. In this work, we introduce tardigrade proteins known for their protective mechanisms into potential LEIA strains to test genetic strategies for improving desiccation and radiation tolerance. Tardigrades are extremotolerant animals well-known for their ability to survive in harsh environments. Notably, they can survive extreme desiccation and radiation due to the upregulation of intrinsically disordered proteins (IDPs). It has been shown that integration of these tardigrade IDPs, such as cytosolic abundant heat-soluble (CAHS) proteins, into yeast can confer increased desiccation tolerance in synergy with trehalose. Our ongoing testing and assessment of bioengineered radiation and desiccation tolerance will help us gain insight into optimizing strain design and selection for protecting LEIA’s biological payload as well as genetic engineering strategies for enhancing bioproduction potential for future missions beyond low Earth orbit.
Genetics↗
Dynamic Implications of Noncovalent Interactions in Amphiphilic Single-Chain Polymer Nanoparticles
Single-chain polymer nanoparticles (SCNPs) combine the chemical diversity of synthetic polymers with the intricate structure of biopolymers, generating versatile biomimetic materials. The mobility of polymer chain segments at length scales similar to secondary structural elements in proteins is critical to SCNP structure and thus function. However, the influence of noncovalent interactions used to form SCNPs (e.g., hydrogen-bonding and biomimetic secondary-like structure) on these conformational dynamics is challenging to quantitatively assess. To isolate the effects of noncovalent interactions on SCNP structure and conformational dynamics, we synthesized a series of amphiphilic copolymers containing dimethylacrylamide and monomers capable of forming these different interactions: (1) di(phenylalanine) acrylamide that forms intramolecular β-sheet-like cross-links, (2) phenylalanine acrylamide that forms hydrogen-bonds but lacks a defined local structure, and (3) benzyl acrylamide that has the lowest propensity for hydrogen-bonding. Each SCNP formed folded structures comparable to those of intrinsically disordered proteins, as observed by size exclusion chromatography and small angle neutron scattering. The dynamics of these polymers, as characterized by a combination of dynamic light scattering and neutron spin echo spectroscopy, was well described using the Zimm with internal friction (ZIF) model, highlighting the role of each noncovalent interaction to additively restrict the internal relaxations of SCNPs. These results demonstrate the utility of local scale interactions to control SCNP polymer dynamics, guiding the design of functional biomimetic materials with refined binding sites and tunable kinetics.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
SAXS of murine amelogenin identifies a persistent dimeric species from pH 5.0 to 8.0
Amelogenin is an intrinsically disordered protein essential to tooth enamel formation in mammals. Here, using advanced small angle X-ray scattering (SAXS) capabilities at synchrotrons and computational models, we revisited measuring the quaternary structure of murine amelogenin as a function of pH and phosphorylation at serine-16. The SAXS data shows that at the pH extremes, amelogenin exists as an extended monomer at pH 3.0 (R g = 38.4 Å) and nanospheres at pH 8.0 (R g = 84.0 Å), consistent with multiple previous observations. At pH 5.0 and above there was no evidence for a significant population of monomeric species. Instead, at pH 5.0 ~ 80% of the population is a heterogenous dimeric species that increases to ~ 100% at pH 5.5. The dimer population was observed at all pH > 5 conditions in dynamic equilibrium with a species in the pentamer range at pH < 6.5 and nanospheres at pH 8.0. At pH 8.0 ~ 40% of the amelogenin still remained in the dimeric state. In general, serine-16 phosphorylation of amelogenin appears to modestly stabilize the population of the dimeric species.
59 BASIC BIOLOGICAL SCIENCES↗
Selective Detection of Intermediate-Amplitude Motion by Solid-State NMR
The coexistence of rigid and mobile molecules or molecular segments abounds in biomolecular assemblies. Examples include the carbohydrate-rich cell walls of plants and intrinsically disordered proteins that contain rigid β-sheet cores. In solid-state nuclear magnetic resonance (NMR) spectroscopy, dipolar polarization transfer experiments are well suited for detecting rigid components, whereas scalar-coupling experiments are well suited for detecting highly mobile components. However, few NMR methods are available to detect the segments that undergo intermediate-amplitude fast motion. Here, we introduce two NMR experiments, a two-dimensional T 2H -filtered CP-hCH correlation and a three-dimensional J-INADEQUATE CCH correlation, to observe this intermediate-amplitude motion. Both experiments involve 1 H detection under fast magic-angle spinning (MAS). By combining 1 H transverse relaxation (T 2H ) filters with dipolar polarization transfer, we suppress the signals of both highly rigid and highly mobile species, thus revealing the signals of intermediate mobile species. 1 H detection under fast MAS is crucial for distinguishing the different motional amplitudes. Here, we demonstrate these techniques on several plant cell wall samples and show that they allow the selective detection and resolution of certain hemicellulose and pectin signals, which are usually masked by the signals of the rigid cellulose and the highly dynamic pectins in purely dipolar and scalar NMR spectra.
59 BASIC BIOLOGICAL SCIENCES↗
Using Multiple Isotope-Labeled Infrared Spectra for the Structural Characterization of an Intrinsically Disordered Peptide
Intrinsically disordered proteins (IDPs) rapidly interconvert between conformers, requiring an ensemble description. This complicates their experimental characterization, and force field limitations pose challenges for their simulation. Here, in this work, we use isotope-labeled and unlabeled infrared (IR) spectra to reweight simulated ensembles of the elastin-like peptide GVGVPGVG, a paradigmatic disordered peptide. By comparing the results obtained with different spectra, we explicitly show that the weights are underdetermined by the ensemble averaged data. We identify which labels and frequency regions maximize structural information while minimizing sensitivity to simulation error and show that these regions report on whether the peptide makes specific interactions. Our work shows the importance of incorporating simulations and simulated spectra at the planning stages of isotope-labeled IR experiments and more generally provides a framework for interpreting IR data for IDPs.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
Role of pore dilation in molecular transport through the nuclear pore complex: Insights from polymer scaling theory
The nuclear pore complex (NPC), a channel within the nuclear envelope filled with intrinsically disordered proteins, regulates the transport of macromolecules between the nucleus and the cytoplasm. Recent studies have highlighted the NPC’s ability to adjust its diameter in response to the membrane tension, underscoring the importance of exploring how variations in pore size influence molecular transport through the NPC. In this study, we investigated the relationship between pore size and transport rate and proposed a mathematical model describing this connection. We began by theoretically analyzing how the pore size scales with the characteristic dimensions of the mesh-like structure within the pore. By introducing key assumptions about how the meshwork structure influences molecular diffusion, we derived a mathematical expression for the transport rate based on the size of the pore and the transported molecules. To validate our model, we conducted Brownian dynamics simulations using a coarse-grained representation of the NPC. These simulations, performed across a range of pore sizes, demonstrated strong agreement with our model’s predictions, confirming its accuracy and applicability. Our model is specifically tailored for small-to-medium-sized molecules, approximately 5 nanometers in size, making it relevant to a wide range of transcription factors and signaling molecules. It also extends to molecules with weak and transient interactions with FG-Nups, such as importin-β. By presenting this model formula, our study offers a quantitative framework for analyzing the effects of pore dilation on nucleocytoplasmic transport.
Biochemistry & Molecular Biology↗