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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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

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.

The Human papillomavirus (HPV) causes tumors in part by hijacking the host cell cycle and forcing uncontrolled cellular division. While there are >200 genotypes of HPV, 15 are classified as high-risk and have been shown to transform infected cells and contribute to tumor formation. The remaining low-risk genotypes are not considered oncogenic and result in benign skin lesions. In high-risk HPV, the oncoprotein E7 contributes to the dysregulation of cell cycle regulatory mechanisms. High-risk E7 is phosphorylated in cells at two conserved serine residues by Casein Kinase 2 (CK2) and this phosphorylation event increases binding affinity for cellular proteins such as the tumor suppressor retinoblastoma (pRb). While low-risk E7 possesses similar serine residues, it is phosphorylated to a lesser degree in cells and has decreased binding capabilities. When E7 binding affinity is decreased, it is less able to facilitate complex interactions between proteins and therefore has less capability to dysregulate the cell cycle. By comparing E7 protein sequences from both low- and high-risk HPV variants and using site-directed mutagenesis combined with NMR spectroscopy and cell-based assays, we demonstrate that the presence of two key nonpolar valine residues within the CK2 recognition sequence, present in low-risk E7, reduces serine phosphorylation efficiency relative to high-risk E7. This results in significant loss of the ability of E7 to degrade the retinoblastoma tumor suppressor protein, thus also reducing the ability of E7 to increase cellular proliferation and reduce senescence. This provides additional insight into the differential E7-mediated outcomes when cells are infected with high-risk verses low-risk HPV. Understanding these oncogenic differences may be important to developing targeted treatment options for HPV-induced cancers., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

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, 19 F-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% and 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., (© 2024 The Protein Society.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Aggregation of transthyretin (TTR) is associated with devastating amyloid diseases. Amyloidosis begins with the 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 19 F-nuclear magnetic resonance (NMR) and a highly sensitive trifluoromethyl probe to determine the relative populations of the species formed by the dissociation of a destabilized variant, A25T. The A25T mutation perturbs both the strong dimer and weak dimer-dimer interfaces. A tetramer ⇌ dimer ⇌ monomer (TDM) equilibrium model is proposed to account for concentration- and temperature-dependent population changes. 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, saturation transfer, and transition state theory. Chemical shifts for the dimer and T* species are degenerate for 19 F and methyl probes close to the strong dimer interface, implicating interfacial perturbation as a common structural feature of these destabilized species. All-atom molecular dynamics simulations further suggest more frequent F87 ring flipping on the nanosecond time scale in the A25T dimer than in the native A25T tetramer. Our integrated approach offers quantitative insights into the energy landscape of the dissociation pathway of TTR at neutral pH.

The cyclic AMP response element (CRE) binding protein (CREB) is a transcription factor that contains a 280-residue N-terminal transactivation domain and a basic leucine zipper that mediates interaction with DNA. The transactivation domain comprises three subdomains, the glutamine-rich domains Q1 and Q2 and the kinase inducible activation domain (KID). NMR chemical shifts show that the isolated subdomains are intrinsically disordered but have a propensity to populate local elements of secondary structure. The Q1 and Q2 domains exhibit a propensity for formation of short β-hairpin motifs that function as binding sites for glutamine-rich sequences. These motifs mediate intramolecular interactions between the CREB Q1 and Q2 domains as well as intermolecular interactions with the glutamine-rich Q1 domain of the TATA-box binding protein associated factor 4 (TAF4) subunit of transcription factor IID (TFIID). Using small-angle X-ray scattering, NMR, and single-molecule Förster resonance energy transfer, we show that the Q1, Q2, and KID regions remain dynamically disordered in a full-length CREB transactivation domain (CREB TAD ) construct. The CREB TAD polypeptide chain is largely extended although some compaction is evident in the KID and Q2 domains. Paramagnetic relaxation enhancement reveals transient long-range contacts both within and between the Q1 and Q2 domains while the intervening KID domain is largely devoid of intramolecular interactions. Phosphorylation results in expansion of the KID domain, presumably making it more accessible for binding the CBP/p300 transcriptional coactivators. Our study reveals the complex nature of the interactions within the intrinsically disordered transactivation domain of CREB and provides molecular-level insights into dynamic and transient interactions mediated by the glutamine-rich domains.

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.

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 IC 50 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.

Viruses infect all kingdoms of life; their genomes vary from DNA to RNA and in size from 2kB to 1 MB or more. Viruses frequently employ disordered proteins, that is, protein products of virus genes that do not themselves fold into independent three-dimensional structures, but rather, constitute a versatile molecular toolkit to accomplish a range of functions necessary for viral infection, assembly, and proliferation. Interestingly, disordered proteins have been discovered in almost all viruses so far studied, whether the viral genome consists of DNA or RNA, and whatever the configuration of the viral capsid or other outer covering. In this review, I present a wide-ranging set of stories illustrating the range of functions of IDPs in viruses. The field is rapidly expanding, and I have not tried to include everything. What is included is meant to be a survey of the variety of tasks that viruses accomplish using disordered proteins., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The accumulation of pathogenic protein oligomers and aggregates is associated with several devastating amyloid diseases. 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 protein dynamics influence aggregation propensity. Kinetic intermediates composed of heterogeneous ensembles of oligomers are frequently formed on the aggregation pathway. Characterization of the structure and dynamics of these intermediates 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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The C-terminal region of the tumor suppressor protein p53 contains three domains, nuclear localization signal (NLS), tetramerization domain (TET), and C-terminal regulatory domain (CTD), which are essential for p53 function. Characterization of the structure and interactions of these domains within full-length p53 has been limited by the overall size and flexibility of the p53 tetramer. Using trans -intein splicing, we have generated full-length p53 constructs in which the C-terminal region is isotopically labeled with 15 N for NMR analysis, allowing us to obtain atomic-level information on the C-terminal domains in the context of the full-length protein. Resonances of NLS and CTD residues have narrow linewidths, showing that these regions are largely solvent-exposed and dynamically disordered, whereas resonances from the folded TET are broadened beyond detection. Two regions of the CTD, spanning residues 369-374 and 381-388 and with high lysine content, make dynamic and sequence-independent interactions with DNA in regions that flank the p53 recognition element. The population of DNA-bound states increases as the length of the flanking regions is extended up to approximately 20 base pairs on either side of the recognition element. Acetylation of K372, K373, and K382, using a construct of the transcriptional coactivator CBP containing the TAZ2 and acetyltransferase domains, inhibits interaction of the CTD with DNA. This work provides high-resolution insights into the behavior of the intrinsically disordered C-terminal regions of p53 within the full-length tetramer and the molecular basis by which the CTD mediates DNA binding and specificity.

The earliest events in the folding of a protein are in general poorly understood. We used NMR R 2 relaxation dispersion experiments to study transient local collapse events in the unfolded-state (U) conformational ensemble of apomyoglobin (apoMb). Local residual secondary structure (seen in regions corresponding to the A, D, E, and H helices of the folded protein) is largely unchanged over the pH range of 2.3-2.75, yet a significant pH-dependent increase in the conformational exchange contribution to the R 2 relaxation rate ( R ex ) indicates that transient intramolecular contacts occur on a microsecond to millisecond time scale at pH 2.75. A comparison of 15 N and 13 CO relaxation dispersion data at pH 2.75 for residues in the A, B, G, and H regions, which participate in the earliest folding intermediates, indicates that chain collapse and secondary structure formation are rapid and concomitant. Increasingly stabilizing conditions (lower temperature, higher pH) result in the observation of a relaxation dispersion in the C, CD, and E regions of the protein, which are known to fold at later stages. Mutation of Trp14 in the A-helix region to Ala eliminates conformational exchange throughout the protein, and the mutation of hydrophobic residues in other regions results in the selective inhibition of conformational exchange in the B, G, or H regions. The R 2 dispersion data for WT apoMb at pH 2.75 and 10 °C are best fit to a four-state model ABGH ⇆ AGH ⇆ U ⇆ ABCD that includes on-pathway (AGH and ABGH) and off-pathway (ABCD) transiently folded states, both of which are required to explain the behavior of the mutant proteins. The off-pathway intermediate is destabilized at higher temperatures. Our analysis provides insights into the earliest stages of apoMb folding where the collapsing polypeptide chain samples both productive and nonproductive states with stabilized secondary structure.

Transthyretin (TTR) amyloidosis is associated with tissue deposition of TTR aggregates. TTR aggregation is initiated by dissociation of the native tetramer to form a monomeric intermediate, which locally unfolds and assembles into soluble oligomers and higher-order aggregates. However, a detailed mechanistic understanding requires kinetic and structural characterization of the low population intermediates formed. Here, we show that the monomeric intermediate exchanges with an ensemble of oligomers on the millisecond timescale. This transient and reversible exchange causes broadening of the 19 F resonance of a trifluoromethyl probe coupled to the monomeric intermediate at S85C. We show the 19 F linewidth and R 2 relaxation rate increase with increasing concentration of the oligomer. Furthermore, introduction of 19 F probes at additional TTR sites yielded distinct 19 F chemical shifts for the TTR tetramer and monomer when the trifluoromethyl probe was attached at S100C, located near the same subunit interface as S85C, but not with probes attached at S46C or E63C, which are distant from any interfaces. The 19 F probe at E63C shows that part of the DE loop, which is solvent accessible in the tetramer, becomes more buried in the NMR-visible oligomers. Finally, using backbone amides as probes, we show that parts of the EF helix and H-strand become highly flexible in the otherwise structured monomeric intermediate at acidic pH. We further find that TTR aggregation can be reversed by increasing pH. Taken together, this work provides insights into location-dependent conformational changes in the reversible early steps of a kinetically concerted TTR aggregation pathway., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Protein dynamics involving higher-energy sparsely populated conformational substates are frequently critical for protein function. This study describes the dynamics of the homodimer (p50)2 of the p50 Rel homology region (RHR) of the transcription factor NF-κB, using 13C relaxation dispersion experiments with specifically (13C, 1H)-labeled methyl groups of Ile (δ), Leu and Val. Free (p50)2 is highly dynamic in solution, showing μs-ms relaxation dispersion consistent with exchange between the ground state and higher energy substates. These fluctuations propagate from the DNA-binding loops through the core of the domain. The motions are damped in the presence of κB DNA, but the NMR spectra of the DNA complexes reveal multiple local conformations of the p50 RHR homodimer bound to certain κB DNA sequences. Varying the length and sequence of κB DNA revealed two factors that promote a single bound conformation for the complex: the length of the κB site in the duplex and a symmetrical sequence of guanine nucleotides at both ends of the recognition motif. The dynamic nature of the DNA-binding loops, together with the multiple bound conformations of p50 RHR with certain κB sites, is consistent with variations in the transcriptional activity of the p50 homodimer with different κB sequences., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The transcription factor NF-κB is one of the central mediators of cellular signaling pathways. Under resting conditions, the canonical RelA-p50 (p65-p50) heterodimer NF-κB remains sequestered in the cytoplasm in complex with its inhibitor IκBα. Signal-mediated activation of NF-κB involves phosphorylation, ubiquitination and degradation of IκBα, and translocation of NF-κB to the nucleus. It was recently shown that a long noncoding RNA (termed NKILA) can modulate the NF-κB signaling circuit by interacting with the NF-κB-IκBα complex in the cytoplasm. In the current study, we investigated the interaction of RNA sequences derived from NKILA with domains of NF-κB and IκBα using NMR spectroscopy and native gel electrophoresis. Our results indicate that two RNA hairpin sequences interact with the DNA-binding domains of the Rel homology regions of RelA (p65) and p50 and that the same RNA sequences can affect the phosphorylation of the N-terminus of IκBα under low-salt conditions. We also observe that full-length RHR dimers (heterodimer of p65 and p50 and homodimer of p50) show a stronger interaction with the RNA hairpins than the individual domains of NF-κB. All of the interactions we observe between fragments of NKILA and domains of NF-κB are weak and nonspecific, consistent with the proposed function of the NKILA-NF-κB-IκBα interaction in protecting the NFκB-IκBα complex from aberrant activation of the NF-κB signaling pathway.

Intrinsically disordered proteins must compete for binding to common regulatory targets to carry out their biological functions. Previously, we showed that the activation domains of two disordered proteins, the transcription factor HIF-1α and its negative regulator CITED2, function as a unidirectional, allosteric molecular switch to control transcription of critical adaptive genes under conditions of oxygen deprivation. These proteins achieve transcriptional control by competing for binding to the TAZ1 domain of the transcriptional coactivators CREB-binding protein (CBP) and p300 (CREB: cyclic-AMP response element binding protein). To characterize the mechanistic details behind this molecular switch, we used solution NMR spectroscopy and complementary biophysical methods to determine the contributions of individual binding motifs in CITED2 to the overall competition process. An N-terminal region of the CITED2 activation domain, which forms a helix when bound to TAZ1, plays a critical role in initiating competition with HIF-1α by enabling formation of a ternary complex in a process that is highly dependent on the dynamics and disorder of the competing partners. Two other conserved binding motifs in CITED2, the LPEL motif and an aromatic/hydrophobic motif that we term ϕC, function synergistically to enhance binding of CITED2 and inhibit rebinding of HIF-1α. The apparent unidirectionality of competition between HIF-1α and CITED2 is lost when one or more of these binding regions is altered by truncation or mutation of the CITED2 peptide. Our findings illustrate the complexity of molecular interactions involving disordered proteins containing multivalent interaction motifs and provide insight into the unique mechanisms by which disordered proteins compete for occupancy of common molecular targets within the cell., Competing Interests: The authors declare no competing interest., (Copyright © 2022 the Author(s). Published by PNAS.)

The intrinsically disordered N-terminal region of the E7 protein from high-risk human papillomavirus (HPV) strains is responsible for oncogenic transformation of host cells through its interaction with a number of cellular factors, including the TAZ2 domain of the transcriptional coactivator CREB-binding protein. Using a variety of spectroscopic and biochemical tools, we find that despite its nanomolar affinity, the HPV16 E7 complex with TAZ2 is disordered and highly dynamic. The disordered domain of HPV16 E7 protein does not adopt a single conformation on the surface of TAZ2 but engages promiscuously with its target through multiple interactions involving two conserved motifs, termed CR1 and CR2, that occupy an extensive binding surface on TAZ2. The fuzzy nature of the complex is a reflection of the promiscuous binding repertoire of viral proteins, which must efficiently dysregulate host cell processes by binding to a variety of host factors in the cellular environment.

Facilitated dissociation provides a mechanism by which high-affinity complexes can be rapidly disassembled. The negative feedback regulator CITED2 efficiently downregulates the hypoxic response by displacing the hypoxia-inducible transcription factor HIF-1α from the TAZ1 domain of the transcriptional coactivators CREB-binding protein (CBP) and p300. Displacement occurs by a facilitated dissociation mechanism involving a transient ternary intermediate formed by binding of the intrinsically disordered CITED2 activation domain to the TAZ1:HIF-1α complex. The short lifetime of the intermediate precludes straightforward structural investigations. To obtain insights into the molecular determinants of facilitated dissociation, we model the ternary intermediate by generating a fusion peptide composed of the primary CITED2 and HIF-1α binding motifs. X-ray crystallographic and NMR studies of the fusion peptide complex reveal TAZ1-mediated negative cooperativity that results in nearly mutually exclusive binding of specific CITED2 and HIF-1α interaction motifs, providing molecular-level insights into the allosteric switch that terminates the hypoxic response., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The study of protein dynamics using the measurement of relaxation times by NMR was based on a set of studies in the mid-20th century that outlined theories and methods. However, the complexity of protein NMR was such that these simple experiments were not practical for application to proteins. The advent of techniques in the 1980s for isotopic labeling of proteins meant that pulse sequences could now be applied in multidimensional NMR experiments to enable per-residue information about the local relaxation times. One of the earliest advances was published in Biochemistry in 1989. The paper "Backbone dynamics of proteins as studied by 15 N inverse detected heteronuclear NMR spectroscopy: application to staphylococcal nuclease" by Lewis Kay, Dennis Torchia, and Ad Bax delineated a set of pulse sequences that are used with minor modifications even today. This paper, with others from a limited number of other laboratories, forms the basis for the experimental determination of the backbone dynamics of proteins. The biological insights obtained from such measurements have only increased in the past 30 years. Sometimes, the best and perhaps only way to advance a field is an advancement in the technical capabilities that allows new perspectives to be reached.

Nuclear magnetic resonance (NMR) has long been instrumental in the characterization of intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs). This method continues to offer rich insights into the nature of IDPs in solution, especially in combination with other biophysical methods such as small-angle scattering, single-molecule fluorescence, electron paramagnetic resonance (EPR), and mass spectrometry. Substantial advances have been made in recent years in studies of proteins containing both ordered and disordered domains and in the characterization of problematic sequences containing repeated tracts of a single or a few amino acids. These sequences are relevant to disease states such as Alzheimer's, Parkinson's, and Huntington's diseases, where disordered proteins misfold into harmful amyloid. Innovative applications of NMR are providing novel insights into mechanisms of protein aggregation and the complexity of IDP interactions with their targets. As a basis for understanding the solution structural ensembles, dynamic behavior, and functional mechanisms of IDPs and IDRs, NMR continues to prove invaluable., Competing Interests: Conflict of interest statement Nothing declared., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Conformational fluctuations from ground-state to sparsely populated but functionally important excited states play a key role in enzyme catalysis. For Escherichia coli dihydrofolate reductase (DHFR), the release of the product tetrahydrofolate (THF) and oxidized cofactor NADP + occurs through exchange between closed and occluded conformations of the Met20 loop. A "dynamic knockout" mutant of E. coli DHFR, where the E. coli sequence in the Met20 loop is replaced by the human sequence (N23PP/S148A), models human DHFR and is incapable of accessing the occluded conformation. 1 H and 15 N CPMG relaxation dispersion analysis for the ternary product complex of the mutant enzyme with NADP + and the product analogue 5,10-dideazatetrahydrofolate (ddTHF) (E:ddTHF:NADP + ) reveals the mechanism by which NADP + is released when the Met20 loop cannot undergo the closed-to-occluded conformational transition. Two excited states were observed: one related to a faster, relatively high-amplitude conformational fluctuation in areas near the active site, associated with the shuttling of the nicotinamide ring of the cofactor out of the active site, and the other to a slower process where ddTHF undergoes small-amplitude motions within the binding site that are consistent with disorder observed in a room-temperature X-ray crystal structure of the N23PP/S148A mutant protein. These motions likely arise due to steric conflict of the pterin ring of ddTHF with the ribose-nicotinamide moiety of NADP + in the closed active site. These studies demonstrate that site-specific kinetic information from relaxation dispersion experiments can provide intimate details of the changes in catalytic mechanism that result from small changes in local amino acid sequence.

Nuclear Factor κB (NF-κB) is a family of five related transcription factors that recognize a κB DNA element on the promoter and enhancer regions of target genes and modulate their expression. Here we report a complete set of 1 H, 13 C, 15 N backbone and side chain resonance assignments for the p50 DNA binding and dimerization domains of the p50 homodimer form of the NF-κB transcription factor. The chemical shift data constitute a first step towards understanding the mechanism of interaction of the p50 homodimer with DNA.

Misfolding and aggregation of transthyretin (TTR) are linked to amyloid disease. Amyloidosis occurs when the TTR homotetramer dissociates into aggregation-prone monomers that self-assemble into amyloid. In familial transthyretin amyloidosis, hereditary amino acid substitutions destabilize TTR and promote aggregation. In this work, we used 19 F nuclear magnetic resonance (NMR) to determine the effect of mutations in the EF helix (Y78F, K80D, K80E, and A81T) and EF loop (G83R and I84S) on the aggregation kinetics and stability of the TTR tetramer and monomer. The EF region acts as a scaffold that stabilizes interactions in both the strong and weak dimer interfaces of the tetramer and is the site of a cluster of pathogenic mutations. K80D and K80E are non-natural mutants that destabilize the EF helix and yield an equilibrium mixture of tetramer and monomer at neutral pH, providing a unique opportunity to determine the thermodynamic parameters for tetramer assembly under nondenaturing conditions. Of the pathogenic mutants studied, only A81T formed appreciable monomer at neutral pH. Real-time 19 F NMR measurements showed that the pathogenic Y78F mutation accelerates aggregation by destabilizing both the tetrameric and monomeric species. The pathogenic mutations A81T, G83R, and I84S destabilize the monomer and increase its aggregation rate by disrupting a Schellman helix C-capping motif. These studies provide new insights into the mechanism by which relatively subtle mutations that affect tetramer or monomer stability promote entry of TTR into the dissociation-aggregation pathway.

We present the first NMR study of the interaction between heat shock protein 90 (Hsp90) and amino (N)-terminal inhibitors 17-AAG, and AUY922, and carboxy (C)-terminal modulators SM253, and LB51. We show that the two ATP mimics, 17-AAG and AUY922, bind deeply within the ATP binding pocket of the N-terminal domain, consistent with the crystal structures. In contrast, SM253, a C-terminal Hsp90 modulator, binds to the linker region between the N and middle domains. We also show that C-terminal inhibitor LB51 binds to the C-terminus with a more significant spectroscopic change than previously reported using NMR binding studies of C-terminal inhibitors novobiocin and silybin. These data provide key insights into how the allosteric inhibitor SM253 controls the C-terminal co-chaperones and confirms the binding domain of LB51., (This journal is © The Royal Society of Chemistry 2021.)

NMR relaxation dispersion measurements report on conformational changes occurring on the μs-ms timescale. Chemical shift information derived from relaxation dispersion can be used to generate structural models of weakly populated alternative conformational states. Current methods to obtain such models rely on determining the signs of chemical shift changes between the conformational states, which are difficult to obtain in many situations. Here, we use a "sample and select" method to generate relevant structural models of alternative conformations of the C-terminal-associated region of Escherichia coli dihydrofolate reductase (DHFR), using only unsigned chemical shift changes for backbone amides and carbonyls ( 1 H, 15 N, and 13 C'). We find that CS-Rosetta sampling with unsigned chemical shift changes generates a diversity of structures that are sufficient to characterize a minor conformational state of the C-terminal region of DHFR. The excited state differs from the ground state by a change in secondary structure, consistent with previous predictions from chemical shift hypersurfaces and validated by the x-ray structure of a partially humanized mutant of E. coli DHFR (N23PP/G51PEKN). The results demonstrate that the combination of fragment modeling with sparse chemical shift data can determine the structure of an alternative conformation of DHFR sampled on the μs-ms timescale. Such methods will be useful for characterizing alternative states, which can potentially be used for in silico drug screening, as well as contributing to understanding the role of minor states in biology and molecular evolution., (Copyright © 2020 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The tumor-suppressor p53 is a critical regulator of the cellular response to DNA damage and is tightly regulated by posttranslational modifications. Thr55 in the AD2 interaction motif of the N-terminal transactivation domain functions as a phosphorylation-dependent regulatory switch that modulates p53 activity. Thr55 is constitutively phosphorylated, becomes dephosphorylated upon DNA damage, and is subsequently rephosphorylated to facilitate dissociation of p53 from promoters and inactivate p53-mediated transcription. Using NMR and fluorescence spectroscopy, we show that Thr55 phosphorylation inhibits DNA-binding by enhancing competitive interactions between the disordered AD2 motif and the structured DNA-binding domain (DBD). Nonphosphorylated p53 exhibits positive cooperativity in binding DNA as a tetramer. Upon phosphorylation of Thr55, cooperativity is abolished and p53 binds initially to cognate DNA sites as a dimer. As the concentration of phosphorylated p53 is further increased, a second dimer binds and causes p53 to dissociate from the DNA, resulting in a bell-shaped binding curve. This autoinhibition is driven by favorable interactions between the DNA-binding surface of the DBD and the multiple phosphorylated AD2 motifs within the tetramer. These interactions are augmented by additional phosphorylation of Ser46 and are fine-tuned by the proline-rich domain (PRD). Removal of the PRD strengthens the AD2-DBD interaction and leads to autoinhibition of DNA binding even in the absence of Thr55 phosphorylation. This study reveals the molecular mechanism by which the phosphorylation status of Thr55 modulates DNA binding and controls both activation and termination of p53-mediated transcriptional programs at different stages of the cellular DNA damage response., Competing Interests: The authors declare no competing interest.

Wilms' tumor suppressor protein WT1 regulates the expression of multiple genes through binding of the Cys 2 -His 2 zinc finger domain to promoter sites. WT1 has also been proposed to be involved in post-transcriptional regulation, by binding to RNA using the same set of zinc fingers. WT1 has two major splice variants, where the Lys-Thr-Ser (KTS) tripeptide is inserted into the linker between the third and fourth zinc fingers. To obtain insights into the mechanism by which the different WT1 splice variants recognize both DNA and RNA, we have determined the solution structure of the WT1 (-KTS) zinc finger domain in complex with a 29mer stem-loop RNA. Zinc fingers 1-3 bind in a widened major groove favored by the presence of a bulge nucleotide in the double-stranded helical stem. Fingers 2 and 3 make specific contacts with the nucleobases in a conserved AUGG sequence in the helical stem. Nuclear magnetic resonance chemical shift mapping and relaxation analysis show that fingers 1-3 of the two splice variants (-KTS and +KTS) of WT1 form similar complexes with RNA. Finger 4 of the -KTS isoform interacts weakly with the RNA loop, an interaction that is abrogated in the +KTS isoform, and both isoforms bind with similar affinity to the RNA. In contrast, finger 4 is required for high-affinity binding to DNA and insertion of KTS into the linker of fingers 3 and 4 abrogates DNA binding. While finger 1 is required for RNA binding, it is dispensable for binding to consensus DNA sites.

Recognition of the epigenetic mark 5-methylcytosine (mC) at CpG sites in DNA has emerged as a novel function of many eukaryotic transcription factors (TFs). It remains unclear why the sequence specificity of these TFs differs for CpG-methylated motifs and consensus motifs. Here, we dissect the structural and dynamic basis for this differential DNA binding specificity in the human zinc finger TF Kaiso, which exhibits high affinity for two consecutive mCpG sites in variable contexts and also for a longer, sequence-specific Kaiso binding site (KBS). By integrating structural analysis and DNA binding studies with targeted protein mutagenesis and nucleotide substitutions, we identify distinct mechanisms for readout of methylated and KBS motifs by Kaiso. We show that a key glutamate residue (E535), critical for mCpG site recognition, adopts different conformations in complexes with specific and methylated DNA. These conformational differences, together with intrinsic variations in DNA flexibility and/or solvation at TpG versus mCpG sites, contribute to the different DNA affinity and sequence specificity. With methylated DNA, multiple direct contacts between E535 and the 5' mCpG site dominate the binding affinity, allowing for tolerance of different flanking DNA sequences. With KBS, Kaiso employs E535 as part of an indirect screen of the 5' flanking sequence, relying on key tyrosine-DNA interactions to stabilize an optimal DNA conformation and select against noncognate sites. These findings demonstrate how TFs use conformational adaptation and exploit variations in DNA flexibility to achieve distinct DNA readout outcomes and target a greater variety of regulatory and epigenetic sites than previously appreciated.

Hsp90 plays an important role in health and is a therapeutic target for managing misfolding disease. Compounds that disrupt co-chaperone delivery of clients to Hsp90 target a subset of Hsp90 activities, thereby minimizing the toxicity of pan-Hsp90 inhibitors. Here, we have identified SEW04784 as a first-in-class inhibitor of the Aha1-stimulated Hsp90 ATPase activity without inhibiting basal Hsp90 ATPase. Nuclear magnetic resonance analysis reveals that SEW84 binds to the C-terminal domain of Aha1 to weaken its asymmetric binding to Hsp90. Consistent with this observation, SEW84 blocks Aha1-dependent Hsp90 chaperoning activities, including the in vitro and in vivo refolding of firefly luciferase, and the transcriptional activity of the androgen receptor in cell-based models of prostate cancer and promotes the clearance of phosphorylated tau in cellular and tissue models of neurodegenerative tauopathy. We propose that SEW84 provides a novel lead scaffold for developing therapeutic approaches to treat proteostatic disease., Competing Interests: Declaration of Interests A provisional patent (docket no. 1361.254PRV) on SEW84 and its derivatives has been filed by Scripps Research on behalf of J.K.S., B.T., and W.E.B. Patents 8933087 and 9201073 are held on IU1, IU1-47 for USP14 inhibition, filed by Harvard University on behalf of D.F. and others. IU1 and IU1-47 patents have been licensed to Proteostasis Therapeutics., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The inactive state of mitogen-activated protein kinases (MAPKs) adopts an open conformation while the active state exists in a compact form stabilized by phosphorylation. In the active state, eukaryotic kinases undergo breathing motions related to substrate binding and product release that have not previously been detected in the inactive state. However, docking interactions of partner proteins with inactive MAPK kinases exhibit allostery in binding of activating kinases. Interactions at a site distant from the activation loop are coupled to the configuration of the activation loop, suggesting that the inactive state may also undergo concerted dynamics. X-ray crystallographic studies of nonphosphorylated, inactive p38γ reveal differences in domain orientations and active site structure in the two molecules in the asymmetric unit. One molecule resembles an inactive kinase with an open active site. The second molecule has a rotation of the N-lobe that leads to partial compaction of the active site, resulting in a conformation that is intermediate between the inactive open state and the fully closed state of the activated kinase. Although the compact state of apo p38γ displays several of the features of the activated enzyme, it remains catalytically inert. In solution, the kinase fluctuates on a millisecond time scale between the open ground state and a weakly populated excited state that is similar in structure to the compact state observed in the crystal. The nuclear magnetic resonance and crystal structure data imply that interconversion between the open and compact states involves a molecular switch associated with the DFG loop.

The nuclear factor of kappa light polypeptide gene enhancer in B-cells (NFκB) transcription factors play a critical role in human immune response. The family includes homodimers and heterodimers of five component proteins, which mediate different transcriptional responses and bind preferentially to different DNA sequences. Crystal structures of DNA complexes show that the dimers of the Rel-homology regions are structurally very similar. Differing DNA sequence preference together with structural similarity suggests that the dimers may differ in their dynamics. In this study, we present the first near-complete 15 N, 13 C α/β , and H N backbone resonance assignments of two dimers of the dimerization domain (DD) of the NFκB1 (p50) protein (residues 241-351): the homodimer of two p50 domains and a heterodimer of the p50 DD with the p65 DD. As expected, the two dimers behave very similarly, with chemical shift differences between them largely concentrated in the dimer interface and attributable to specific differences in the amino acid sequences of p50 and p65. A comparison of the picosecond-nanosecond dynamics of the homo- and heterodimers also shows that the environment of p50 is similar, with an overall slightly reduced correlation time for the homodimer compared to the heterodimer, consistent with its slightly smaller molecular weight. These results demonstrate that NMR spectroscopy can be used to explore subtle changes in structure and dynamics that have the potential to give insights into differences in specificity that can be exploited in the design of new therapeutic agents., (© 2019 The Protein Society.)

The 2019 ISMAR Prize recognized NMR studies of disordered proteins. Here we provide a highly personal perspective on the discovery of intrinsically disordered proteins and the development and application of NMR methods to characterize their conformational ensembles, dynamics, and interactions.

The structured DNA-binding domain (DBD) of p53 is a well-known client protein of the chaperone Hsp90. The p53 DBD contains a single zinc ion, coordinated by the side chains of Cys176, His179, Cys238, and Cys242; zinc coordination plays a structural role to stabilize the DBD and is required for its DNA binding. The ambiguous nature of the p53-Hsp90 interaction, together with the stabilizing role of the zinc in the structure of the DBD, prompted us to examine the interaction of Hsp90 with zinc-free p53 DBD. NMR spectroscopy and native gel electrophoresis did not show any apparent preference for the interaction of the destabilized zinc-free form of p53 DBD with Hsp90. Intriguingly, however, at lower protein concentrations, closer to physiological concentrations, the addition of Hsp90, but not other chaperones such as Hsp70, Hsp40, p23, and HOP, appears to slow or prevent the aggregation of zinc-free p53 DBD. This result suggests that part of the function of the Hsp90-p53 interaction in the cell may be to stabilize the apoprotein in the absence of zinc., (© 2019 The Protein Society.)

The intrinsically disordered transactivation domains of HIF-1α and CITED2 compete for binding of the TAZ1 domain of the CREB-binding protein by a unidirectional allosteric mechanism involving direct competition for shared binding sites, ternary complex formation, and TAZ1 conformational changes. To gain insight into the mechanism by which CITED2 displaces HIF-1α from TAZ1, we used nuclear magnetic resonance spin relaxation methods to obtain an atomic-level description of the picosecond to nanosecond backbone dynamics that contribute to TAZ1 binding and competition. We show that HIF-1α and CITED2 adopt different dynamics in their complexes with TAZ1, with flexibility observed for HIF-1α in regions that would maintain accessibility for CITED2 to bind to TAZ1 and facilitate subsequent HIF-1α dissociation. In contrast, critical regions of CITED2 adopt a rigid structure in its complex with TAZ1, minimizing the ability of HIF-1α to compete for binding. We also find that TAZ1, previously thought to be a rigid scaffold for binding of disordered protein ligands, displays altered backbone dynamics in its various bound states. TAZ1 is more rigid in its CITED2-bound state than in its free state or in complex with HIF-1α, with increased rigidity observed not only in the CITED2 binding site but also in regions of TAZ1 that undergo conformational changes between the HIF-1α- and CITED2-bound structures. Taken together, these data suggest that backbone dynamics in TAZ1, as well as in the HIF-1α and CITED2 ligands, play a role in modulating the occupancy of TAZ1 and highlight the importance of characterizing both binding partners in molecular interactions.

Phosphorylation of the kinase inducible domain (KID) of the cyclic AMP response element binding transcription factor (CREB) regulates its function through several mechanisms. Transcriptional activation occurs following phosphorylation at serine 133, but multisite phosphorylation in a neighboring region termed the CK cassette, residues 108-117, results in inhibition of CREB-mediated transcription. A molecular-level understanding of the mechanism of these opposing reactions has been lacking, in part because of the difficulty of preparing multiply phosphorylated CREB in vitro. By substituting a single residue, we have generated an engineered mammalian CREB in which the CK cassette can be phosphorylated in vitro by casein kinases and have characterized its interactions with cyclic AMP response element DNA. Phosphorylation of the CK cassette promotes an intramolecular interaction between the KID domain and the site of DNA binding, the basic region of the C-terminal basic leucine zipper (bZip) domain. Competition between the phosphorylated KID domain and DNA for bZip binding results in a decreased affinity of CREB for DNA. The binding free energy calculated from the dissociation constant is directly proportional to the number of phosphate groups in the CK cassette, indicating that the DNA binding is regulated by a rheostat-like mechanism. The rheostat is modulated by variation of the concentration of cations such as Mg 2+ and by alternative isoforms such as the natural CREB isoform that lacks residues 162-272. Multisite phosphorylation of CREB represents a versatile mechanism by which transcription can be tuned to meet the variable needs of the cell.

Aggregation of transthyretin (TTR) causes TTR amyloidoses. The native TTR tetramer (a dimer of dimers) is stabilized by packing of phenylalanine 87 (F87) into a hydrophobic cavity of a neighboring protomer across the strong dimer interface. X-ray structures at acidic pH show that the side chain of F87 can be displaced from its binding pocket, but the resultant solution conformations remain unknown. Here we used 19 F nuclear magnetic resonance (NMR) and 19 F-labeled C10S-S85C TTR to characterize two local conformations of the loop containing F87. At neutral pH, F87 packs correctly into the interprotomer cavity in the dominant conformational state (93% population, T) whereas the remaining minor population is a mispacked tetramer (T*). The population of T* can be enhanced in heterotetramers by mixing C10S-S85C TTR with increasing molar ratios of A120L TTR, where a bulky leucine residue is introduced to disfavor the T state by steric hindrance. Exchange between the T and T* states in the presence of A120L is mediated by subunit exchange from the C10S-S85C tetramer. Compared to the TTR tetramer in which the dimers are correctly packed, mispacking of one or both dimer pairs leads to an increase in the urea unfolding rate of 4-fold or at least 15-fold, respectively. Consistent acid-mediated tetramer dissociation was observed by 19 F NMR aggregation assays. Our results highlight the important role of the interprotomer F87 side chain packing in determining the kinetic stability of the TTR tetramer; mispacking of F87 in the T* state predisposes it for rapid dissociation and entry into the aggregation pathway.

Atomic resolution characterization of the full-length p53 tetramer has been hampered by its size and the presence of extensive intrinsically disordered regions at both the N and C termini. As a consequence, the structural characteristics and dynamics of the disordered regions are poorly understood within the context of the intact p53 tetramer. Here we apply trans -intein splicing to generate segmentally 15 N-labeled full-length p53 constructs in which only the resonances of the N-terminal transactivation domain (NTAD) are visible in NMR spectra, allowing us to observe this region of p53 with unprecedented detail within the tetramer. The N-terminal region is dynamically disordered in the full-length p53 tetramer, fluctuating between states in which it is free and fully exposed to solvent and states in which it makes transient contacts with the DNA-binding domain (DBD). Chemical-shift changes and paramagnetic spin-labeling experiments reveal that the amphipathic AD1 and AD2 motifs of the NTAD interact with the DNA-binding surface of the DBD through primarily electrostatic interactions. Importantly, this interaction inhibits binding of nonspecific DNA to the DBD while having no effect on binding to a specific p53 recognition element. We conclude that the NTAD:DBD interaction functions to enhance selectivity toward target genes by inhibiting binding to nonspecific sites in genomic DNA. This work provides some of the highest-resolution data on the disordered N terminus of the nearly 180-kDa full-length p53 tetramer and demonstrates a regulatory mechanism by which the N terminus of p53 transiently interacts with the DBD to enhance target site discrimination., Competing Interests: The authors declare no conflict of interest.

The human T cell leukemia virus I basic leucine zipper protein (HTLV-1 HBZ) maintains chronic viral infection and promotes leukemogenesis through poorly understood mechanisms involving interactions with the KIX domain of the transcriptional coactivator CBP and its paralog p300. The KIX domain binds regulatory proteins at the distinct MLL and c-Myb/pKID sites to form binary or ternary complexes. The intrinsically disordered N-terminal activation domain of HBZ (HBZ AD) deregulates cellular signaling pathways by competing directly with cellular and viral transcription factors for binding to the MLL site and by allosterically perturbing binding of the transactivation domain of the hematopoietic transcription factor c-Myb. Crystal structures of the ternary KIX:c-Myb:HBZ complex show that the HBZ AD recruits two KIX:c-Myb entities through tandem amphipathic motifs (L/V)(V/L)DGLL and folds into a long α-helix upon binding. Isothermal titration calorimetry reveals strong cooperativity in binding of the c-Myb activation domain to the KIX:HBZ complex and in binding of HBZ to the KIX:c-Myb complex. In addition, binding of KIX to the two HBZ (V/L)DGLL motifs is cooperative; the structures suggest that this cooperativity is achieved through propagation of the HBZ α-helix beyond the first binding motif. Our study suggests that the unique structural flexibility and the multiple interaction motifs of the intrinsically disordered HBZ AD are responsible for its potency in hijacking KIX-mediated transcription pathways. The KIX:c-Myb:HBZ complex provides an example of cooperative stabilization in a transcription factor:coactivator network and gives insights into potential mechanisms through which HBZ dysregulates hematopoietic transcriptional programs and promotes T cell proliferation., Competing Interests: The authors declare no conflict of interest.

Allosteric regulatory processes are implicated at all levels of biological function. Recent advances in our understanding of the diverse and functionally significant class of intrinsically disordered proteins have identified a multitude of ways in which disordered proteins function within the confines of the allosteric paradigm. Allostery within or mediated by intrinsically disordered proteins ensures robust and efficient signal integration through mechanisms that would be extremely unfavorable or even impossible for globular protein interaction partners. Here, we highlight recent examples that indicate the breadth of biological outcomes that can be achieved through allosteric regulation by intrinsically disordered proteins. Ongoing and future work in this rapidly evolving area of research will expand our appreciation of the central role of intrinsically disordered proteins in ensuring the fidelity and efficiency of cellular regulation., (Copyright © 2018 Elsevier Ltd. All rights reserved.)