Intrinsically disordered domain of transcription factor TCF-1 is required for T cell developmental fidelity.

In development, pioneer transcription factors access silent chromatin to reveal lineage-specific gene programs. The structured DNA-binding domains of pioneer factors have been well characterized, but whether and how intrinsically disordered regions affect chromatin and control cell fate is unclear. Here, we report that deletion of an intrinsically disordered region of the pioneer factor TCF-1 (termed L1) leads to an early developmental block in T cells. The few T cells that develop from progenitors expressing TCF-1 lacking L1 exhibit lineage infidelity distinct from the lineage diversion of TCF-1-deficient cells. Mechanistically, L1 is required for activation of T cell genes and repression of GATA2-driven genes, normally reserved to the mast cell and dendritic cell lineages. Underlying this lineage diversion, L1 mediates binding of TCF-1 to its earliest target genes, which are subject to repression as T cells develop. These data suggest that the intrinsically disordered N terminus of TCF-1 maintains T cell lineage fidelity.

Mechanisms of inhibition of human monoclonal antibodies in immune thrombotic thrombocytopenic purpura.

Antibody binding to a plasma metalloprotease, a disintegrin and metalloproteinase with thrombospondin type 1 repeats 13 (ADAMTS13), is necessary for the development of immune thrombotic thrombocytopenic purpura (iTTP). Inhibition of ADAMTS13-mediated von Willebrand factor (VWF) cleavage by such antibodies clearly plays a role in the pathophysiology of the disease, although the mechanisms by which they inhibit ADAMTS13 enzymatic function are not fully understood. At least some immunoglobulin G-type antibodies appear to affect the conformational accessibility of ADAMTS13 domains involved in both substrate recognition and inhibitory antibody binding. We used single-chain fragments of the variable region previously identified via phage display from patients with iTTP to explore the mechanisms of action of inhibitory human monoclonal antibodies. Using recombinant full-length ADAMTS13, truncated ADAMTS13 variants, and native ADAMTS13 in normal human plasma, we found that, regardless of the conditions tested, all 3 inhibitory monoclonal antibodies tested affected enzyme turnover rate much more than substrate recognition of VWF. Hydrogen-to-deuterium exchange plus mass spectrometry experiments with each of these inhibitory antibodies demonstrated that residues in the active site of the catalytic domain of ADAMTS13 are differentially exposed to solvent in the presence and absence of monoclonal antibody binding. These results support the hypothesis that inhibition of ADAMTS13 in iTTP may not necessarily occur because the antibodies directly prevent VWF binding, but instead because of allosteric effects that impair VWF cleavage, likely by affecting the conformation of the catalytic center in the protease domain of ADAMTS13. Our findings provide novel insight into the mechanism of autoantibody-mediated inhibition of ADAMTS13 and pathogenesis of iTTP.

Structural and kinetic basis for the regulation and potentiation of Hsp104 function.

Hsp104 provides a valuable model for the many essential proteostatic functions performed by the AAA+ superfamily of protein molecular machines. We developed and used a powerful hydrogen exchange mass spectrometry (HX MS) analysis that can provide positionally resolved information on structure, dynamics, and energetics of the Hsp104 molecular machinery, even during functional cycling. HX MS reveals that the ATPase cycle is rate-limited by ADP release from nucleotide-binding domain 1 (NBD1). The middle domain (MD) serves to regulate Hsp104 activity by slowing ADP release. Mutational potentiation accelerates ADP release, thereby increasing ATPase activity. It reduces time in the open state, thereby decreasing substrate protein loss. During active cycling, Hsp104 transits repeatedly between whole hexamer closed and open states. Under diverse conditions, the shift of open/closed balance can lead to premature substrate loss, normal processing, or the generation of a strong pulling force. HX MS exposes the mechanisms of these functions at near-residue resolution.

A conserved strategy for structure change and energy transduction in Hsp104 and other AAA+ protein motors.

The superfamily of massively large AAA+ protein molecular machines functions to convert the chemical energy of cytosolic ATP into physicomechanical form and use it to perform an extraordinary number of physical operations on proteins, nucleic acids, and membrane systems. Cryo-EM studies now reveal some aspects of substrate handling at high resolution, but the broader interpretation of AAA+ functional properties is still opaque. This paper integrates recent hydrogen exchange results for the typical AAA+ protein Hsp104 with prior information on several near and distantly related others. The analysis points to a widely conserved functional strategy. Hsp104 cycles through a long-lived loosely-structured energy-input "open" state that releases spent ADP and rebinds cytosolic ATP. ATP-binding energy is transduced by allosteric structure change to poise the protein at a high energy level in a more tightly structured "closed" state. The briefly occupied energy-output closed state binds substrate strongly and is catalytically active. ATP hydrolysis permits energetically downhill structural relaxation, which is coupled to drive energy-requiring substrate processing. Other AAA+ proteins appear to cycle through states that are analogous functionally if not in structural detail. These results revise the current model for AAA+ function, explain the structural basis of single-molecule optical tweezer kinetic phases, identify the separate energetic roles of ATP binding and hydrolysis, and specify a sequence of structural and energetic events that carry AAA+ proteins unidirectionally around a functional cycle to propel their diverse physical tasks.

Recommendations for performing, interpreting and reporting hydrogen deuterium exchange mass spectrometry (HDX-MS) experiments.

Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a powerful biophysical technique being increasingly applied to a wide variety of problems. As the HDX-MS community continues to grow, adoption of best practices in data collection, analysis, presentation and interpretation will greatly enhance the accessibility of this technique to nonspecialists. Here we provide recommendations arising from community discussions emerging out of the first International Conference on Hydrogen-Exchange Mass Spectrometry (IC-HDX; 2017). It is meant to represent both a consensus viewpoint and an opportunity to stimulate further additions and refinements as the field advances.

Hydrogen exchange reveals Hsp104 architecture, structural dynamics, and energetics in physiological solution.

Hsp104 is a large AAA+ molecular machine that can rescue proteins trapped in amorphous aggregates and stable amyloids by drawing substrate protein into its central pore. Recent cryo-EM studies image Hsp104 at high resolution. We used hydrogen exchange mass spectrometry analysis (HX MS) to resolve and characterize all of the functionally active and inactive elements of Hsp104, many not accessible to cryo-EM. At a global level, HX MS confirms the one noncanonical interprotomer interface in the Hsp104 hexamer as a marker for the spiraled conformation revealed by cryo-EM and measures its fast conformational cycling under ATP hydrolysis. Other findings enable reinterpretation of the apparent variability of the regulatory middle domain. With respect to detailed mechanism, HX MS determines the response of each Hsp104 structural element to the different bound adenosine nucleotides (ADP, ATP, AMPPNP, and ATPγS). They are distinguished most sensitively by the two Walker A nucleotide-binding segments. Binding of the ATP analog, ATPγS, tightly restructures the Walker A segments and drives the global open-to-closed/extended transition. The global transition carries part of the ATP/ATPγS-binding energy to the somewhat distant central pore. The pore constricts and the tyrosine and other pore-related loops become more tightly structured, which seems to reflect the energy-requiring directional pull that translocates the substrate protein. ATP hydrolysis to ADP allows Hsp104 to relax back to its lowest energy open state ready to restart the cycle.

Structural and mechanistic insights into Hsp104 function revealed by synchrotron X-ray footprinting.

Hsp104 is a hexameric AAA+ ring translocase, which drives protein disaggregation in nonmetazoan eukaryotes. Cryo-EM structures of Hsp104 have suggested potential mechanisms of substrate translocation, but precisely how Hsp104 hexamers disaggregate proteins remains incompletely understood. Here, we employed synchrotron X-ray footprinting to probe the solution-state structures of Hsp104 monomers in the absence of nucleotide and Hsp104 hexamers in the presence of ADP or ATPγS (adenosine 5'-O-(thiotriphosphate)). Comparing side-chain solvent accessibilities between these three states illuminated aspects of Hsp104 structure and guided design of Hsp104 variants to probe the disaggregase mechanism in vitro and in vivo We established that Hsp104 hexamers switch from a more-solvated state in ADP to a less-solvated state in ATPγS, consistent with switching from an open spiral to a closed ring visualized by cryo-EM. We pinpointed critical N-terminal domain (NTD), NTD-nucleotide-binding domain 1 (NBD1) linker, NBD1, and middle domain (MD) residues that enable intrinsic disaggregase activity and Hsp70 collaboration. We uncovered NTD residues in the loop between helices A1 and A2 that can be substituted to enhance disaggregase activity. We elucidated a novel potentiated Hsp104 MD variant, Hsp104-RYD, which suppresses α-synuclein, fused in sarcoma (FUS), and TDP-43 toxicity. We disambiguated a secondary pore-loop in NBD1, which collaborates with the NTD and NBD1 tyrosine-bearing pore-loop to drive protein disaggregation. Finally, we defined Leu-601 in NBD2 as crucial for Hsp104 hexamerization. Collectively, our findings unveil new facets of Hsp104 structure and mechanism. They also connect regions undergoing large changes in solvation to functionality, which could have profound implications for protein engineering.

Folding of maltose binding protein outside of and in GroEL.

We used hydrogen exchange-mass spectrometry (HX MS) and fluorescence to compare the folding of maltose binding protein (MBP) in free solution and in the GroEL/ES cavity. Upon refolding, MBP initially collapses into a dynamic molten globule-like ensemble, then forms an obligatory on-pathway native-like folding intermediate (1.2 seconds) that brings together sequentially remote segments and then folds globally after a long delay (30 seconds). A single valine to glycine mutation imposes a definable folding defect, slows early intermediate formation by 20-fold, and therefore subsequent global folding by approximately twofold. Simple encapsulation within GroEL repairs the folding defect and reestablishes fast folding, with or without ATP-driven cycling. Further examination exposes the structural mechanism. The early folding intermediate is stabilized by an organized cluster of 24 hydrophobic side chains. The cluster preexists in the collapsed ensemble before the H-bond formation seen by HX MS. The V9G mutation slows folding by disrupting the preintermediate cluster. GroEL restores wild-type folding rates by restabilizing the preintermediate, perhaps by a nonspecific equilibrium compression effect within its tightly confining central cavity. These results reveal an active GroEL function other than previously proposed mechanisms, suggesting that GroEL possesses different functionalities that are able to relieve different folding problems. The discovery of the preintermediate, its mutational destabilization, and its restoration by GroEL encapsulation was made possible by the measurement of a previously unexpected type of low-level HX protection, apparently not dependent on H-bonding, that may be characteristic of proteins in confined spaces.

The case for defined protein folding pathways.

We consider the differences between the many-pathway protein folding model derived from theoretical energy landscape considerations and the defined-pathway model derived from experiment. A basic tenet of the energy landscape model is that proteins fold through many heterogeneous pathways by way of amino acid-level dynamics biased toward selecting native-like interactions. The many pathways imagined in the model are not observed in the structure-formation stage of folding by experiments that would have found them, but they have now been detected and characterized for one protein in the initial prenucleation stage. Analysis presented here shows that these many microscopic trajectories are not distinct in any functionally significant way, and they have neither the structural information nor the biased energetics needed to select native vs. nonnative interactions during folding. The opposed defined-pathway model stems from experimental results that show that proteins are assemblies of small cooperative units called foldons and that a number of proteins fold in a reproducible pathway one foldon unit at a time. Thus, the same foldon interactions that encode the native structure of any given protein also naturally encode its particular foldon-based folding pathway, and they collectively sum to produce the energy bias toward native interactions that is necessary for efficient folding. Available information suggests that quantized native structure and stepwise folding coevolved in ancient repeat proteins and were retained as a functional pair due to their utility for solving the difficult protein folding problem.

Helical structure, stability, and dynamics in human apolipoprotein E3 and E4 by hydrogen exchange and mass spectrometry.

Apolipoprotein E (apoE) plays a critical role in cholesterol transport in both peripheral circulation and brain. Human apoE is a polymorphic 299-residue protein in which the less common E4 isoform differs from the major E3 isoform only by a C112R substitution. ApoE4 interacts with lipoprotein particles and with the amyloid-ß peptide, and it is associated with increased incidence of cardiovascular and Alzheimer's disease. To understand the structural basis for the differences between apoE3 and E4 functionality, we used hydrogen-deuterium exchange coupled with a fragment separation method and mass spectrometric analysis to compare their secondary structures at near amino acid resolution. We determined the positions, dynamics, and stabilities of the helical segments in these two proteins, in their normal tetrameric state and in mutation-induced monomeric mutants. Consistent with prior X-ray crystallography and NMR results, the N-terminal domain contains four α-helices, 20 to 30 amino acids long. The C-terminal domain is relatively unstructured in the monomeric state but forms an α-helix ∼70 residues long in the self-associated tetrameric state. Helix stabilities are relatively low, 4 kcal/mol to 5 kcal/mol, consistent with flexibility and facile reversible unfolding. Secondary structure in the tetrameric apoE3 and E4 isoforms is similar except that some helical segments in apoE4 spanning residues 12 to 20 and 204 to 210 are unfolded. These conformational differences result from the C112R substitution in the N-terminal helix bundle and likely relate to a reduced ability of apoE4 to form tetramers, thereby increasing the concentration of functional apoE4 monomers, which gives rise to its higher lipid binding compared with apoE3.

ExMS2: An Integrated Solution for Hydrogen-Deuterium Exchange Mass Spectrometry Data Analysis.

Hydrogen-deuterium exchange mass spectrometry (HDX MS) has become an important technique for the analysis of protein structure and dynamics. Data analysis remains a bottleneck in the workflow. Sophisticated computer analysis is required to scan through the voluminous MS output in order to find, identify, and validate many partially deuterated peptides, elicit the HDX information, and extend the results to higher structural resolution. We previously made available two software suites, ExMS for identification and analysis of peptide isotopic envelopes in the HDX MS raw data and HDsite for residue-level resolution. Further experience has led to advances in the usability and performance of both programs. Also, newly added modules deal with ETD/ECD analysis, multimodal mass spectra analysis, and presentation options. These advances have been integrated into a stand-alone software solution named ExMS2. The package has been successfully tested by many workers in fine scale epitope mapping, in protein folding studies, and in dissecting structure and structure change of large protein complexes. A description and tutorial for this major upgrade are given here.

Cytochrome c folds through foldon-dependent native-like intermediates in an ordered pathway.

Previous hydrogen exchange (HX) studies of the spontaneous reversible unfolding of Cytochrome c (Cyt c) under native conditions have led to the following conclusions. Native Cyt c (104 residues) is composed of five cooperative folding units, called foldons. The high-energy landscape is dominated by an energy ladder of partially folded forms that differ from each other by one cooperative foldon unit. The reversible equilibrium unfolding of native Cyt c steps up through these intermediate forms to the unfolded state in an energy-ordered sequence, one foldon unit at a time. To more directly study Cyt c intermediates and pathways during normal energetically downhill kinetic folding, the present work used HX pulse labeling analyzed by a fragment separation-mass spectrometry method. The results show that 95% or more of the Cyt c population folds by stepping down through the same set of foldon-dependent pathway intermediates as in energetically uphill equilibrium unfolding. These results add to growing evidence that proteins fold through a classical pathway sequence of native-like intermediates rather than through a vast number of undefinable intermediates and pathways. The present results also emphasize the condition-dependent nature of kinetic barriers, which, with less informative experimental methods (fluorescence, etc.), are often confused with variability in intermediates and pathways.

High-resolution epitope mapping by HX MS reveals the pathogenic mechanism and a possible therapy for autoimmune TTP syndrome.

Acquired thrombotic thrombocytopenic purpura (TTP), a thrombotic disorder that is fatal in almost all cases if not treated promptly, is primarily caused by IgG-type autoantibodies that inhibit the ability of the ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) metalloprotease to cleave von Willebrand factor (VWF). Because the mechanism of autoantibody-mediated inhibition of ADAMTS13 activity is not known, the only effective therapy so far is repeated whole-body plasma exchange. We used hydrogen-deuterium exchange mass spectrometry (HX MS) to determine the ADAMTS13 binding epitope for three representative human monoclonal autoantibodies, isolated from TTP patients by phage display as tethered single-chain fragments of the variable regions (scFvs). All three scFvs bind the same conformationally discontinuous epitopic region on five small solvent-exposed loops in the spacer domain of ADAMTS13. The same epitopic region is also bound by most polyclonal IgG autoantibodies in 23 TTP patients that we tested. The ability of ADAMTS13 to proteolyze VWF is impaired by the binding of autoantibodies at the epitopic loops in the spacer domain, by the deletion of individual epitopic loops, and by some local mutations. Structural considerations and HX MS results rule out any disruptive structure change effect in the distant ADAMTS13 metalloprotease domain. Instead, it appears that the same ADAMTS13 loop segments that bind the autoantibodies are also responsible for correct binding to the VWF substrate. If so, the autoantibodies must prevent VWF proteolysis simply by physically blocking normal ADAMTS13 to VWF interaction. These results point to the mechanism for autoantibody action and an avenue for therapeutic intervention.

The nature of protein folding pathways.

How do proteins fold, and why do they fold in that way? This Perspective integrates earlier and more recent advances over the 50-y history of the protein folding problem, emphasizing unambiguously clear structural information. Experimental results show that, contrary to prior belief, proteins are multistate rather than two-state objects. They are composed of separately cooperative foldon building blocks that can be seen to repeatedly unfold and refold as units even under native conditions. Similarly, foldons are lost as units when proteins are destabilized to produce partially unfolded equilibrium molten globules. In kinetic folding, the inherently cooperative nature of foldons predisposes the thermally driven amino acid-level search to form an initial foldon and subsequent foldons in later assisted searches. The small size of foldon units, ∼ 20 residues, resolves the Levinthal time-scale search problem. These microscopic-level search processes can be identified with the disordered multitrack search envisioned in the "new view" model for protein folding. Emergent macroscopic foldon-foldon interactions then collectively provide the structural guidance and free energy bias for the ordered addition of foldons in a stepwise pathway that sequentially builds the native protein. These conclusions reconcile the seemingly opposed new view and defined pathway models; the two models account for different stages of the protein folding process. Additionally, these observations answer the "how" and the "why" questions. The protein folding pathway depends on the same foldon units and foldon-foldon interactions that construct the native structure.

Protein hydrogen exchange at residue resolution by proteolytic fragmentation mass spectrometry analysis.

Hydrogen exchange technology provides a uniquely powerful instrument for measuring protein structural and biophysical properties, quantitatively and in a nonperturbing way, and determining how these properties are implemented to produce protein function. A developing hydrogen exchange-mass spectrometry method (HX MS) is able to analyze large biologically important protein systems while requiring only minuscule amounts of experimental material. The major remaining deficiency of the HX MS method is the inability to deconvolve HX results to individual amino acid residue resolution. To pursue this goal we used an iterative optimization program (HDsite) that integrates recent progress in multiple peptide acquisition together with previously unexamined isotopic envelope-shape information and a site-resolved back-exchange correction. To test this approach, residue-resolved HX rates computed from HX MS data were compared with extensive HX NMR measurements, and analogous comparisons were made in simulation trials. These tests found excellent agreement and revealed the important computational determinants.

Folding of a large protein at high structural resolution.

Kinetic folding of the large two-domain maltose binding protein (MBP; 370 residues) was studied at high structural resolution by an advanced hydrogen-exchange pulse-labeling mass-spectrometry method (HX MS). Dilution into folding conditions initiates a fast molecular collapse into a polyglobular conformation (<20 ms), determined by various methods including small angle X-ray scattering. The compaction produces a structurally heterogeneous state with widespread low-level HX protection and spectroscopic signals that match the equilibrium melting posttransition-state baseline. In a much slower step (7-s time constant), all of the MBP molecules, although initially heterogeneously structured, form the same distinct helix plus sheet folding intermediate with the same time constant. The intermediate is composed of segments that are distant in the MBP sequence but adjacent in the native protein where they close the longest residue-to-residue contact. Segments that are most HX protected in the early molecular collapse do not contribute to the initial intermediate, whereas the segments that do participate are among the less protected. The 7-s intermediate persists through the rest of the folding process. It contains the sites of three previously reported destabilizing mutations that greatly slow folding. These results indicate that the intermediate is an obligatory step on the MBP folding pathway. MBP then folds to the native state on a longer time scale (~100 s), suggestively in more than one step, the first of which forms structure adjacent to the 7-s intermediate. These results add a large protein to the list of proteins known to fold through distinct native-like intermediates in distinct pathways.

Stepwise protein folding at near amino acid resolution by hydrogen exchange and mass spectrometry.

The kinetic folding of ribonuclease H was studied by hydrogen exchange (HX) pulse labeling with analysis by an advanced fragment separation mass spectrometry technology. The results show that folding proceeds through distinct intermediates in a stepwise pathway that sequentially incorporates cooperative native-like structural elements to build the native protein. Each step is seen as a concerted transition of one or more segments from an HX-unprotected to an HX-protected state. Deconvolution of the data to near amino acid resolution shows that each step corresponds to the folding of a secondary structural element of the native protein, termed a "foldon." Each folded segment is retained through subsequent steps of foldon addition, revealing a stepwise buildup of the native structure via a single dominant pathway. Analysis of the pertinent literature suggests that this model is consistent with experimental results for many proteins and some current theoretical results. Two biophysical principles appear to dictate this behavior. The principle of cooperativity determines the central role of native-like foldon units. An interaction principle termed "sequential stabilization" based on native-like interfoldon interactions orders the pathway.

Apolipoprotein A-I helical structure and stability in discoidal high-density lipoprotein (HDL) particles by hydrogen exchange and mass spectrometry.

To understand high-density lipoprotein (HDL) structure at the molecular level, the location and stability of α-helical segments in human apolipoprotein (apo) A-I in large (9.6 nm) and small (7.8 nm) discoidal HDL particles were determined by hydrogen-deuterium exchange (HX) and mass spectrometry methods. The measured HX kinetics of some 100 apoA-I peptides specify, at close to amino acid resolution, the structural condition of segments throughout the protein sequence and changes in structure and stability that occur on incorporation into lipoprotein particles. When incorporated into the large HDL particle, the nonhelical regions in lipid-free apoA-I (residues 45-53, 66-69, 116-146, and 179-236) change conformation from random coil to α-helix so that nearly the entire apoA-I molecule adopts helical structure (except for the terminal residues 1-6 and 237-243). The amphipathic α-helices have relatively low stability, in the range 3-5 kcal/mol, indicating high flexibility and dynamic unfolding and refolding in seconds or less. A segment encompassed by residues 125-158 exhibits bimodal HX labeling indicating co-existing helical and disordered loop conformations that interchange on a time scale of minutes. When incorporated around the edge of the smaller HDL particle, the increase in packing density of the two apoA-I molecules forces about 20% more residues out of direct contact with the phospholipid molecules to form disordered loops, and these are the same segments that form loops in the lipid-free state. The region of disc-associated apoA-I that binds the lecithin-cholesterol acyltransferase enzyme is well structured and not a protruding unstructured loop as reported by others.