The transthyretin (TTR) tetramer, assembled as a dimer of dimers, transports thyroxine and retinol binding protein in blood plasma and cerebrospinal fluid. Aggregation of wild type (WT) or pathogenic variant TTR leads to transthyretin amyloidosis, which is associated with neurodegenerative and cardiac disease. The trigger for TTR aggregation under physiological conditions is unknown. The tetramer is extremely stable at neutral pH, but aggregation via tetramer dissociation and monomer misfolding can be induced in vitro by lowering the pH. To elucidate factors that may cause TTR aggregation at neutral pH, we examined the effect of shear forces such as those that arise from fluid flow in the vascular system. Fluid shear forces were generated by rapidly stirring TTR solutions in conical microcentrifuge tubes. Under agitation, TTR formed β-rich aggregates and fibrils at a rate that was dependent upon protein concentration. The lag time before the onset of agitation-induced aggregation increases as the total TTR concentration is increased, consistent with a mechanism in which the tetramer first dissociates to form monomer that either partially unfolds to enter the aggregation pathway or reassociates to form tetramer. NMR spectra recorded at various time points during the lag phase revealed growth of an aggregation-prone intermediate trapped as a dynamically perturbed tetramer. Enhanced conformational fluctuations in the weak dimer–dimer interface suggest loosening of critical intersubunit contacts which likely destabilizes the agitated tetramer and predisposes it toward dissociation. These studies provide insights into the mechanism of aggregation of WT human TTR under near-physiological conditions.

The proper assembly of light-harvesting complexes (LHCs) is critical for photosynthesis and requires the biogenesis of light-harvesting chlorophyll a,b-binding proteins (LHCPs) to be coordinated with chlorophyll (Chl) biosynthesis. The mechanism underlying this coordination is not well understood. Here, we show that a conserved molecular chaperone, chloroplast signal recognition particle 43-kDa protein (cpSRP43), provides a molecular thermostat that helps maintain this coordination. cpSRP43 undergoes a conformational rearrangement between a well-folded closed state and a partially disordered open state. Closed cpSRP43 is dedicated to the biogenesis of LHCPs, whereas open cpSRP43 protects multiple Chl biosynthesis enzymes from heat-induced destabilization. Rising temperature shifts cpSRP43 to the open state, enabling it to protect heat-destabilized Chl biosynthesis enzymes. Our results reveal the molecular basis of a posttranslational mechanism for the thermoadaptation of LHC biogenesis. They also demonstrate how an adenosine triphosphate-independent chaperone uses conformational dynamics to switch its activity and client selectivity, thereby adapting to different proteostatic demands under shifting environmental conditions.

Aggregation of transthyretin (TTR) causes TTR cardiomyopathy and polyneuropathy through amyloidosis. To initialize TTR aggregation, the native TTR tetramer first dissociates to a monomeric intermediate, which misfolds and self-assembles to oligomers, eventually forming insoluble aggregates and fibrils. Peptide inhibitors have been designed to cap two β-strands that are buried in the well-folded tetramer but are solvent-exposed in the monomeric aggregation intermediate. However, how these peptides affect the reaction kinetics of individual steps in the multi-step TTR aggregation pathway remains unknown. Here, we integrated 19F-NMR and kinetic modeling to determine aggregation reaction rates of individual steps with the peptide inhibitors and extract the free energy landscape along the TTR aggregation pathway. We found direct kinetic evidence that the peptide inhibitors bind to monomeric intermediates and misfolded tetramers at acidic pH, but do not bind to structured TTR monomers or tetramers at neutral pH. In addition, the peptides do not bind to amorphous aggregates formed at acidic pH and physiological temperature in vitro, in contrast to the previously reported findings that these peptides recognize ex vivo TTR fibrils derived from patients with TTR amyloidosis. Interestingly, the peptides bind to soluble oligomers formed at acidic pH and low temperature in vitro, suggesting that these oligomers may share structural similarity with misfolded monomeric intermediates and patient-derived TTR fibrils. Our methods provide quantitative and mechanistic details for peptide-inhibited TTR aggregation.

Disulfide bond protein A (DsbA) plays a pivotal role in catalysing the formation of disulfide bonds within the periplasm of most Gram-negative bacteria. As this process is required for the folding of multiple virulence-associated proteins, inhibitors of DsbA have the potential to be developed as novel anti-virulence agents. Despite extensive efforts to develop high-affinity inhibitors targeting the canonical DsbA substrate-binding site, success has been limited. In the current work, we describe a dynamics-driven fragment discovery approach. Our analysis of EcDsbA dynamics using nuclear magnetic resonance relaxation-dispersion data revealed a novel “cryptic” ligand binding pocket within Escherichia coli DsbA (EcDsbA). Compounds bind to this cryptic pocket with unusually slow kinetics and a preference for interacting with oxidised EcDsbA, the predominant form of the protein found in the periplasm. We characterise an induced-fit mechanism, involving conformational changes in the active-site helix of EcDsbA, observed preferentially in the oxidised state, which facilitates access to the cryptic pocket. This conformational exchange explains both the slow kinetics and the redox-dependent binding of the ligands. Furthermore, we demonstrate that compounds binding to the cryptic pocket inhibit EcDsbA activity in vitro. These findings highlight the potential of the cryptic pocket as a target site for developing more potent inhibitors of EcDsbA.

Intrinsically disordered proteins that mediate the cellular transcriptional response to hypoxia play important roles in regulating oxygen stress genes. The feedback inhibitor CITED2 operates a unidirectional switch that efficiently terminates the hypoxic response by displacing the C-terminal activation domain of the hypoxia-inducible factor HIF-1α from its complex with the TAZ1 domain of the transcriptional coactivators CBP and p300. Unidirectionality of the switch arises from subtle allosteric conformational changes in TAZ1 and from differences in the strength of thermodynamic coupling between the TAZ1-binding motifs in the multivalent HIF-1α and CITED2 activation domains. To investigate the role of binding cooperativity, we mutated a linker sequence in the HIF-1α activation domain to alter the thermodynamic coupling between its TAZ1-binding motifs. Linker mutations that enhance the affinity of HIF-1α for TAZ1 enable the HIF-1α activation domain to compete more effectively with bound CITED2. The wide range of mutants, which include insertion, deletion, substitution, and scrambling of residues in the linker, provide insights into the molecular basis for the exquisite tuning of the hypoxic switch. The TAZ1 binding affinity and consequent CITED2 competition enhancement depends both on the flexibility of the linker sequence (particularly the presence of glycine residues) and the unfavorable electrostatic interactions of a highly conserved arginine side chain in the center of the linker with an electropositive surface of TAZ1. The conservation of the linker length and sequence in all vertebrates suggests strong evolutionary pressure to tune HIF-1α binding affinity to be sub-optimal, to ensure unidirectionality of the hypoxic switch.

In his commentary in this issue of Molecular Cell,1 Struhl reasons that the term "intrinsically disordered regions" represents a vague and confusing concept for protein function. However, the term "intrinsically disordered" highlights the important physicochemical characteristic of conformational heterogeneity. Thus, "intrinsically disordered" is the counterpart to the term "folded, " with neither term having specific functional implications.

The aggregation pathway of transthyretin (TTR) proceeds through rate-limiting dissociation of the tetramer (a dimer of dimers) and partial misfolding of the resulting monomer, which assembles into amyloid structures through a downhill polymerization mechanism. The structural features of the aggregation-prone monomeric intermediate are poorly understood. NMR relaxation dispersion offers a unique opportunity to characterize amyloidogenic intermediates when they exchange on favorable timescales with NMR-visible ground states. Here we use NMR to characterize the structure and conformational dynamics of the monomeric F87E mutant of human TTR. Chemical shifts derived from analysis of multinuclear relaxation dispersion data provide insights into the structure of a low-lying excited state that exchanges with the ground state of the F87E monomer at a rate of 3800 s-1. Disruption of the subunit interfaces of the TTR tetramer leads to destabilization of edge strands in both β-sheets of the F87E monomer. Conformational fluctuations are propagated through the entire hydrogen bonding network of the DAGH β-sheet, from the inner β-strand H, which forms the strong dimer-dimer interface in the TTR tetramer, to outer strand D which is unfolded in TTR fibrils. Fluctuations are also propagated from the AB loop in the weak dimer-dimer interface to the EF helix, which undergoes structural remodeling in fibrils. The conformational fluctuations in both regions are enhanced at acidic pH where amyloid formation is most favorable. The relaxation dispersion data provide insights into the conformational dynamics of the amyloidogenic state of monomeric TTR that predispose it for structural remodeling and progression to amyloid fibrils.

Defects in the tumor suppressor protein p53 are found in the majority of cancers. The p53 protein (393 amino acids long) contains the folded DNA-binding domain (DBD) and tetramerization domain (TET), with the remainder of the sequence being intrinsically disordered. Since cancer-causing mutations occur primarily in the DBD, this has been the focus of most of the research on p53. However, recent reports show that the disordered N-terminal activation domain (NTAD) and C-terminal regulatory domain (CTD) function synergistically with the DBD to regulate p53 activity. We propose a mechanistic model in which intermolecular and intramolecular interactions of the disordered regions, modulated by post-translational modifications, perform a central role in the regulation and activation of p53 in response to cellular stress.

Aggregation of transthyretin (TTR) is associated with devastating amyloid diseases. Amyloidosis begins with dissociation of the native homotetramer (a dimer of dimers) to form a monomeric intermediate that assembles into pathogenic aggregates. This process is accelerated in vitro at low pH, but the process by which TTR dissociates and reassembles at neutral pH remains poorly characterized due to the low population of intermediates. Here we use 19F NMR and a highly sensitive trifluoromethyl probe to determine the relative populations of the species formed by dissociation of the destabilized A25T variant.

The transthyretin (TTR) tetramer, assembled as a dimer of dimers, transports thyroxine and retinol binding protein in blood plasma and cerebrospinal fluid. Aggregation of wild type or pathogenic variant TTR leads to transthyretin amyloidosis (ATTR), which is associated with neurodegenerative and cardiac disease. The trigger for TTR aggregation under physiological conditions is unknown. The tetramer is extremely stable at neutral pH, but aggregation via tetramer dissociation and monomer misfolding can be induced in vitro by lowering the pH. To elucidate factors that may cause TTR aggregation at neutral pH, we examined the effect of shear forces such as arise from fluid flow in the vascular system. Fluid shear forces were generated by rapidly stirring TTR solutions in conical microcentrifuge tubes. Under agitation, TTR formed β-rich aggregates and fibrils at a rate that was dependent upon protein concentration. The lag time before the onset of agitation-induced aggregation increases as the total TTR concentration is increased, consistent with a mechanism in which the tetramer first dissociates to form monomer that either partially unfolds to enter the aggregation pathway or reassociates to form tetramer. NMR spectra recorded at various time points during the lag phase revealed growth of an aggregation-prone intermediate trapped as a dynamically perturbed tetramer. Enhanced conformational fluctuations in the weak dimer-dimer interface suggests loosening of critical inter-subunit contacts which likely destabilizes the agitated tetramer and predisposes it towards dissociation. These studies provide new insights into the mechanism of aggregation of wild type human TTR under near physiological conditions.

Aberrant formation and deposition of human transthyretin (TTR) aggregates causes transthyretin amyloidosis. To initialize aggregation, transthyretin tetramers must first dissociate into monomers that partially unfold to promote entry into the aggregation pathway. The native TTR tetramer (T) is stabilized by docking of the F87 sidechain into an interfacial cavity enclosed by several hydrophobic residues including A120. We have previously shown that an alternative tetramer (T*) with mispacked F87 sidechains is more prone to dissociation and aggregation than the native T state. However, the molecular basis for the reduced stability in T* remains unclear. Here we report characterization of the A120L mutant, where steric hindrance is introduced into the F87 binding site. The X-ray structure of A120L shows that the F87 sidechain is displaced from its docking site across the subunit interface. In A120S, a naturally occurring pathogenic mutant that is less aggregation-prone than A120L, the F87 sidechain is correctly docked, as in the native TTR tetramer. Nevertheless, 19F-NMR aggregation assays show an elevated population of a monomeric aggregation intermediate in A120S relative to a control containing the native A120, due to accelerated tetramer dissociation and slowed monomer tetramerization. The mispacking of the F87 sidechain is associated with enhanced exchange dynamics for interfacial residues. At 298 K, the T* populations of various naturally occurring mutants fall between 4-7% (ΔG ~ 1.5-1.9 kcal/mol), consistent with the free energy change expected for undocking and solvent exposure of one of the four F87 sidechains in the tetramer (ΔG ~ 1.6 kcal/mol). Our data provide a molecular-level picture of the likely universal F87 sidechain mispacking in tetrameric TTR that promotes interfacial conformational dynamics and increases aggregation propensity.

The aggregation pathway of transthyretin (TTR) proceeds through rate-limiting dissociation of the tetramer and partial misfolding of the monomers, which assemble into amyloid structures through a downhill polymerization mechanism. The structural features of the aggregation-prone monomeric intermediate are poorly understood. Characterization of amyloidogenic intermediates is challenging due to their propensity to aggregate at concentrations necessary for structural studies. NMR relaxation dispersion offers a unique opportunity to characterize these intermediates when they exchange on favorable timescales with NMR-visible ground states. To characterize the structural transitions associated with tetramer dissociation, we have analyzed ground-state chemical shift differences between the native tetramer and an engineered monomer in which the critical F87 side chain is replaced by glutamate. The secondary structure and overall fold of the F87E monomer is similar to that of the tetramer except for β-strand H. This strand populates two conformations, where it is either docked on the protein core or is displaced from the edge of the β-sheet formed by β-strands D, A, G, and H (DAGH β-sheet) and is dynamically disordered. Chemical shift differences derived from analysis of 1H/15N single, double and zero quantum relaxation dispersion data provide insights into the structure of a low-lying excited state that exchanges with the ground state of the F87E monomer at a rate of 3800 s-1. Disruption of the subunit interfaces of the TTR tetramer leads to destabilization of edge strands in both β-sheets of the F87E monomer. Conformational fluctuations are propagated through the entire hydrogen bonding network of the DAGH β-sheet, from the inner β-strand H, which forms the strong dimer interface in the TTR tetramer, to outer strand D which is unfolded in TTR fibrils. Fluctuations are also propagated from the AB loop in the weak dimer interface to the EF helix, which undergoes structural remodeling in fibrils. The conformational fluctuations in both regions are enhanced at acidic pH where amyloid formation is most favorable. The relaxation dispersion data provide insights into the conformational dynamics of the amyloidogenic state of monomeric TTR that predispose it for structural remodeling and progression to amyloid fibrils.### Competing Interest StatementThe authors have declared no competing interest.

The accumulation of pathogenic protein oligomers and ag-gregates is associated with several devastating amyloid dis-eases. As protein aggregation is a multi-step nucleation-dependent process beginning with unfolding or misfolding of the native state, it is important to understand how innate pro-tein dynamics influence aggregation propensity. Kinetic in-ter mediates composed of heterogeneous ensembles of oligomers are frequently formed on the aggregation pathway. Characterization of the structure and dynamics of these in-ter mediates is critical to the understanding of amyloid diseases since oligomers appear to be the main cytotoxic agents. In this review, we highlight recent biophysical studies of the roles of protein dynamics in driving pathogenic protein aggregation, yielding new mechanistic insights that can be leveraged for design of aggregation inhibitors.

It is hard to evaluate the role of individual mentors in the genesis of important ideas. In the case of our realization that proteins do not have to be stably folded to be functional, the influence of Richard Lerner and our collaborative work in the 1980s on the conformations of immunogenic peptides provided a base level of thinking about the nature of polypeptides in water solutions that led us to formulate and develop our ideas on the importance of intrinsic disorder in proteins. This review describes how the insights gained into the behavior of peptides led directly to the realization that proteins were not only capable of being functional while disordered, but also that disorder provided a distinct functional advantage in many important cellular processes.

Aggregation of transthyretin (TTR) is associated with devastating TTR amyloid disease. Amyloidosis begins with dissociation of the native tetramer to form a monomeric intermediate that assembles into pathogenic aggregates. This process is accelerated in vitro at low pH, but the dissociation and reassembly of TTR at neutral pH remains poorly understood, due to the low population of intermediates. We use NMR studies with a highly sensitive 19 F probe that allows deconvolution of relative populations of a destabilized A25T mutant at concentrations as low as 2 µM. The A25T mutation, located at the weak dimer interface, perturbs both the weak and strong dimer interfaces. A tetramer-dimer-monomer (TDM) equilibrium model is proposed to account for concentration- and temperature-dependent population changes. All thermodynamic and kinetic parameters and activation energetics for dissociation of the native A25T tetramer, as well as a destabilized alternative tetramer (T*) with a mispacked F87 side chain, were extracted by van’t Hoff and 19 F NMR line-shape analysis. The conversion from T to T*, the slowest first-order kinetic step, shows anti-Arrhenius behavior. The 19 F and methyl chemical shifts of probes close to the strong dimer interface in the dimer and T* species are degenerate, implicating interfacial perturbation as a common structural feature of these intermediate species. Molecular dynamics (MD) simulations further suggest more frequent F87 ring flipping on the nanoscale timescale in the A25T dimer than in the tetramer. Our integrated approach offers quantitative insights into the energy landscape of the dissociation pathway of TTR at neutral pH.

Cows produce antibodies with a disulfide-bonded antigen-binding domain embedded within ultralong heavy chain third complementarity determining regions. This "knob" domain is analogous to natural cysteine-rich peptides such as knottins in that it is small and stable but can accommodate diverse loops and disulfide bonding patterns. We immunized cattle with SARS-CoV-2 spike and found ultralong CDR H3 antibodies that could neutralize several viral variants at picomolar IC50 potencies in vitro and could protect from disease in vivo. The independent CDR H3 peptide knobs were expressed and maintained the properties of the parent antibodies. The knob interaction with SARS-CoV-2 spike was revealed by electron microscopy, X-ray crystallography, NMR spectroscopy, and mass spectrometry and established ultralong CDR H3-derived knobs as the smallest known recombinant independent antigen-binding fragment. Unlike other vertebrate antibody fragments, these knobs are not reliant on the immunoglobulin domain and have potential as a new class of therapeutics.