Exploring the dynamics and interactions of the N-myc transactivation domain through solution nuclear magnetic resonance spectroscopy.

Myc proteins are transcription factors crucial for cell proliferation. They have a C-terminal domain that mediates Max and DNA binding, and an N-terminal disordered region culminating in the transactivation domain (TAD). The TAD participates in many protein-protein interactions, notably with kinases that promote stability (Aurora-A) or degradation (ERK1, GSK3) via the ubiquitin-proteasome system. We probed the structure, dynamics and interactions of N-myc TAD using nuclear magnetic resonance (NMR) spectroscopy following its complete backbone assignment. Chemical shift analysis revealed that N-myc has two regions with clear helical propensity: Trp77-Glu86 and Ala122-Glu132. These regions also have more restricted ps-ns motions than the rest of the TAD, and, along with the phosphodegron, have comparatively high transverse (R2) 15N relaxation rates, indicative of slower timescale dynamics and/or chemical exchange. Collectively these features suggest differential propensities for structure and interaction, either internal or with binding partners, across the TAD. Solution studies on the interaction between N-myc and Aurora-A revealed a previously uncharacterised binding site. The specificity and kinetics of sequential phosphorylation of N-myc by ERK1 and GSK3 were characterised using NMR and resulted in no significant structural changes outside the phosphodegron. When the phosphodegron was doubly phosphorylated, N-myc formed a robust interaction with the Fbxw7-Skp1 complex, but mapping the interaction by NMR suggests a more extensive interface. Our study provides foundational insights into N-myc TAD dynamics and a backbone assignment that will underpin future work on the structure, dynamics, interactions and regulatory post-translational modifications of this key oncoprotein.

Chaperone BiP controls ER stress sensor Ire1 through interactions with its oligomers.

The complex multistep activation cascade of Ire1 involves changes in the Ire1 conformation and oligomeric state. Ire1 activation enhances ER folding capacity, in part by overexpressing the ER Hsp70 molecular chaperone BiP; in turn, BiP provides tight negative control of Ire1 activation. This study demonstrates that BiP regulates Ire1 activation through a direct interaction with Ire1 oligomers. Particularly, we demonstrated that the binding of Ire1 luminal domain (LD) to unfolded protein substrates not only trigger conformational changes in Ire1-LD that favour the formation of Ire1-LD oligomers but also exposes BiP binding motifs, enabling the molecular chaperone BiP to directly bind to Ire1-LD in an ATP-dependent manner. These transient interactions between BiP and two short motifs in the disordered region of Ire1-LD are reminiscent of interactions between clathrin and another Hsp70, cytoplasmic Hsc70. BiP binding to substrate-bound Ire1-LD oligomers enables unfolded protein substrates and BiP to synergistically and dynamically control Ire1-LD oligomerisation, helping to return Ire1 to its deactivated state when an ER stress response is no longer required.

Intrinsic disorder and conformational coexistence in auxin coreceptors.

AUXIN/INDOLE 3-ACETIC ACID (Aux/IAA) transcriptional repressor proteins and the TRANSPORT INHIBITOR RESISTANT 1/AUXIN SIGNALING F-BOX (TIR1/AFB) proteins to which they bind act as auxin coreceptors. While the structure of TIR1 has been solved, structural characterization of the regions of the Aux/IAA protein responsible for auxin perception has been complicated by their predicted disorder. Here, we use NMR, CD and molecular dynamics simulation to investigate the N-terminal domains of the Aux/IAA protein IAA17/AXR3. We show that despite the conformational flexibility of the region, a critical W-P bond in the core of the Aux/IAA degron motif occurs at a strikingly high (1:1) ratio of cis to trans isomers, consistent with the requirement of the cis conformer for the formation of the fully-docked receptor complex. We show that the N-terminal half of AXR3 is a mixture of multiple transiently structured conformations with a propensity for two predominant and distinct conformational subpopulations within the overall ensemble. These two states were modeled together with the C-terminal PB1 domain to provide the first complete simulation of an Aux/IAA. Using MD to recreate the assembly of each complex in the presence of auxin, both structural arrangements were shown to engage with the TIR1 receptor, and contact maps from the simulations match closely observations of NMR signal-decreases. Together, our results and approach provide a platform for exploring the functional significance of variation in the Aux/IAA coreceptor family and for understanding the role of intrinsic disorder in auxin signal transduction and other signaling systems.

Generating Ensembles of Dynamic Misfolding Proteins.

The early stages of protein misfolding and aggregation involve disordered and partially folded protein conformers that contain a high degree of dynamic disorder. These dynamic species may undergo large-scale intra-molecular motions of intrinsically disordered protein (IDP) precursors, or flexible, low affinity inter-molecular binding in oligomeric assemblies. In both cases, generating atomic level visualization of the interconverting species that captures the conformations explored and their physico-chemical properties remains hugely challenging. How specific sub-ensembles of conformers that are on-pathway to aggregation into amyloid can be identified from their aggregation-resilient counterparts within these large heterogenous pools of rapidly moving molecules represents an additional level of complexity. Here, we describe current experimental and computational approaches designed to capture the dynamic nature of the early stages of protein misfolding and aggregation, and discuss potential challenges in describing these species because of the ensemble averaging of experimental restraints that arise from motions on the millisecond timescale. We give a perspective of how machine learning methods can be used to extract aggregation-relevant sub-ensembles and provide two examples of such an approach in which specific interactions of defined species within the dynamic ensembles of α-synuclein (αSyn) and ß2-microgloblulin (ß2m) can be captured and investigated.

Structural basis of Apt48 inhibition of the BCL6 BTB domain.

B cell lymphoma 6 (BCL6) is a transcriptional repressor that is deregulated in diffuse large B cell lymphoma, and the peptide aptamer, Apt48, inhibits BCL6 by an unknown mechanism. We report the crystal structure of BCL6 in complex with an Apt48 peptide, and show that Apt48 binds to a therapeutically uncharacterized region at the bottom of the BCL6 BTB domain. We show that the corepressor binding site of the BTB domain may be divided conceptually into two low-affinity, peptide-binding regions. An upper region, the lateral groove, binds peptides in robust three-dimensional conformations, whereas a lower binding site is permissive to less-specific interactions. We show that, even with little sequence specificity, the interactions of the lower region are required for the high-affinity binding of the SMRT corepressor and other peptides to the BTB domain. This has relevance for the design of new BCL6 inhibitors and for understanding the evolution of corepressor interactions with the BTB domain.

Grb2 binding induces phosphorylation-independent activation of Shp2.

The regulation of phosphatase activity is fundamental to the control of intracellular signalling and in particular the tyrosine kinase-mediated mitogen-activated protein kinase (MAPK) pathway. Shp2 is a ubiquitously expressed protein tyrosine phosphatase and its kinase-induced hyperactivity is associated with many cancer types. In non-stimulated cells we find that binding of the adaptor protein Grb2, in its monomeric state, initiates Shp2 activity independent of phosphatase phosphorylation. Grb2 forms a bidentate interaction with both the N-terminal SH2 and the catalytic domains of Shp2, releasing the phosphatase from its auto-inhibited conformation. Grb2 typically exists as a dimer in the cytoplasm. However, its monomeric state prevails under basal conditions when it is expressed at low concentration, or when it is constitutively phosphorylated on a specific tyrosine residue (Y160). Thus, Grb2 can activate Shp2 and downstream signal transduction, in the absence of extracellular growth factor stimulation or kinase-activating mutations, in response to defined cellular conditions. Therefore, direct binding of Grb2 activates Shp2 phosphatase in the absence of receptor tyrosine kinase up-regulation.

Mutations in Spliceosomal Genes PPIL1 and PRP17 Cause Neurodegenerative Pontocerebellar Hypoplasia with Microcephaly.

Autosomal-recessive cerebellar hypoplasia and ataxia constitute a group of heterogeneous brain disorders caused by disruption of several fundamental cellular processes. Here, we identified 10 families showing a neurodegenerative condition involving pontocerebellar hypoplasia with microcephaly (PCHM). Patients harbored biallelic mutations in genes encoding the spliceosome components Peptidyl-Prolyl Isomerase Like-1 (PPIL1) or Pre-RNA Processing-17 (PRP17). Mouse knockouts of either gene were lethal in early embryogenesis, whereas PPIL1 patient mutation knockin mice showed neuron-specific apoptosis. Loss of either protein affected splicing integrity, predominantly affecting short and high GC-content introns and genes involved in brain disorders. PPIL1 and PRP17 form an active isomerase-substrate interaction, but we found that isomerase activity is not critical for function. Thus, we establish disrupted splicing integrity and "major spliceosome-opathies" as a new mechanism underlying PCHM and neurodegeneration and uncover a non-enzymatic function of a spliceosomal proline isomerase.

Fusidic acid resistance through changes in the dynamics of the drug target.

Antibiotic resistance in clinically important bacteria can be mediated by target protection mechanisms, whereby a protein binds to the drug target and protects it from the inhibitory effects of the antibiotic. The most prevalent source of clinical resistance to the antibiotic fusidic acid (FA) is expression of the FusB family of proteins that bind to the drug target (Elongation factor G [EF-G]) and promote dissociation of EF-G from FA-stalled ribosome complexes. FusB binding causes changes in both the structure and conformational flexibility of EF-G, but which of these changes drives FA resistance was not understood. We present here detailed characterization of changes in the conformational flexibility of EF-G in response to FusB binding and show that these changes are responsible for conferring FA resistance. Binding of FusB to EF-G causes a significant change in the dynamics of domain III of EF-GC3 that leads to an increase in a minor, more disordered state of EF-G domain III. This is sufficient to overcome the steric block of transmission of conformational changes within EF-G by which FA prevents release of EF-G from the ribosome. This study has identified an antibiotic resistance mechanism mediated by allosteric effects on the dynamics of the drug target.

Crystal structure of the putative cyclase IdmH from the indanomycin nonribosomal peptide synthase/polyketide synthase.

Indanomycin is biosynthesized by a hybrid nonribosomal peptide synthase/polyketide synthase (NRPS/PKS) followed by a number of 'tailoring' steps to form the two ring systems that are present in the mature product. It had previously been hypothesized that the indane ring of indanomycin was formed by the action of IdmH using a Diels-Alder reaction. Here, the crystal structure of a selenomethionine-labelled truncated form of IdmH (IdmH-Δ99-107) was solved using single-wavelength anomalous dispersion (SAD) phasing. This truncated variant allows consistent and easy crystallization, but importantly the structure was used as a search model in molecular replacement, allowing the full-length IdmH structure to be determined to 2.7 Šresolution. IdmH is a homodimer, with the individual protomers consisting of an α+ß barrel. Each protomer contains a deep hydrophobic pocket which is proposed to constitute the active site of the enzyme. To investigate the reaction catalysed by IdmH, 88% of the backbone NMR resonances were assigned, and using chemical shift perturbation of [15N]-labelled IdmH it was demonstrated that indanomycin binds in the active-site pocket. Finally, combined quantum mechanical/molecular mechanical (QM/MM) modelling of the IdmH reaction shows that the active site of the enzyme provides an appropriate environment to promote indane-ring formation, supporting the assignment of IdmH as the key Diels-Alderase catalysing the final step in the biosynthesis of indanomycin through a similar mechanism to other recently characterized Diels-Alderases involved in polyketide-tailoring reactions. An animated Interactive 3D Complement (I3DC) is available in Proteopedia at https://proteopedia.org/w/Journal:IUCrJ:S2052252519012399.

Structural mapping of oligomeric intermediates in an amyloid assembly pathway.

Transient oligomers are commonly formed in the early stages of amyloid assembly. Determining the structure(s) of these species and defining their role(s) in assembly is key to devising new routes to control disease. Here, using a combination of chemical kinetics, NMR spectroscopy and other biophysical methods, we identify and structurally characterize the oligomers required for amyloid assembly of the protein ΔN6, a truncation variant of human ß2-microglobulin (ß2m) found in amyloid deposits in the joints of patients with dialysis-related amyloidosis. The results reveal an assembly pathway which is initiated by the formation of head-to-head non-toxic dimers and hexamers en route to amyloid fibrils. Comparison with inhibitory dimers shows that precise subunit organization determines amyloid assembly, while dynamics in the C-terminal strand hint to the initiation of cross-ß structure formation. The results provide a detailed structural view of early amyloid assembly involving structured species that are not cytotoxic.

Dynamic ion pair behavior stabilizes single α-helices in proteins.

Ion pairs are key stabilizing interactions between oppositely charged amino acid side chains in proteins. They are often depicted as single conformer salt bridges (hydrogen-bonded ion pairs) in crystal structures, but it is unclear how dynamic they are in solution. Ion pairs are thought to be particularly important in stabilizing single α-helix (SAH) domains in solution. These highly stable domains are rich in charged residues (such as Arg, Lys, and Glu) with potential ion pairs across adjacent turns of the helix. They provide a good model system to investigate how ion pairs can contribute to protein stability. Using NMR spectroscopy, small-angle X-ray light scattering (SAXS), and molecular dynamics simulations, we provide here experimental evidence that ion pairs exist in a SAH in murine myosin 7a (residues 858-935), but that they are not fixed or long lasting. In silico modeling revealed that the ion pairs within this α-helix exhibit dynamic behavior, rapidly forming and breaking and alternating between different partner residues. The low-energy helical state was compatible with a great variety of ion pair combinations. Flexible ion pair formation utilizing a subset of those available at any one time avoided the entropic penalty of fixing side chain conformations, which likely contributed to helix stability overall. These results indicate the dynamic nature of ion pairs in SAHs. More broadly, thermodynamic stability in other proteins is likely to benefit from the dynamic behavior of multi-option solvent-exposed ion pairs.

Protein Surface Mimetics: Understanding How Ruthenium Tris(Bipyridines) Interact with Proteins.

Protein surface mimetics achieve high-affinity binding by exploiting a scaffold to project binding groups over a large area of solvent-exposed protein surface to make multiple cooperative noncovalent interactions. Such recognition is a prerequisite for competitive/orthosteric inhibition of protein-protein interactions (PPIs). This paper describes biophysical and structural studies on ruthenium(II) tris(bipyridine) surface mimetics that recognize cytochrome (cyt) c and inhibit the cyt c/cyt c peroxidase (CCP) PPI. Binding is electrostatically driven, with enhanced affinity achieved through enthalpic contributions thought to arise from the ability of the surface mimetics to make a greater number of noncovalent interactions than CCP with surface-exposed basic residues on cyt c. High-field natural abundance 1 H,15 N HSQC NMR experiments are consistent with surface mimetics binding to cyt c in similar manner to CCP. This provides a framework for understanding recognition of proteins by supramolecular receptors and informing the design of ligands superior to the protein partners upon which they are inspired.

A Population Shift between Sparsely Populated Folding Intermediates Determines Amyloidogenicity.

The balance between protein folding and misfolding is a crucial determinant of amyloid assembly. Transient intermediates that are sparsely populated during protein folding have been identified as key players in amyloid aggregation. However, due to their ephemeral nature, structural characterization of these species remains challenging. Here, using the power of nonuniformly sampled NMR methods we investigate the folding pathway of amyloidogenic and nonamyloidogenic variants of ß2-microglobulin (ß2m) in atomic detail. Despite folding via common intermediate states, we show that the decreased population of the aggregation-prone ITrans state and population of a less stable, more dynamic species ablate amyloid formation by increasing the energy barrier for amyloid assembly. The results show that subtle changes in conformational dynamics can have a dramatic effect in determining whether a protein is amyloidogenic, without perturbation of the mechanism of protein folding.

A target-protection mechanism of antibiotic resistance at atomic resolution: insights into FusB-type fusidic acid resistance.

Antibiotic resistance in clinically important bacteria can be mediated by proteins that physically associate with the drug target and act to protect it from the inhibitory effects of an antibiotic. We present here the first detailed structural characterization of such a target protection mechanism mediated through a protein-protein interaction, revealing the architecture of the complex formed between the FusB fusidic acid resistance protein and the drug target (EF-G) it acts to protect. Binding of FusB to EF-G induces conformational and dynamic changes in the latter, shedding light on the molecular mechanism of fusidic acid resistance.

Mechanisms of amyloid formation revealed by solution NMR.

Amyloid fibrils are proteinaceous elongated aggregates involved in more than fifty human diseases. Recent advances in electron microscopy and solid state NMR have allowed the characterization of fibril structures to different extents of refinement. However, structural details about the mechanism of fibril formation remain relatively poorly defined. This is mainly due to the complex, heterogeneous and transient nature of the species responsible for assembly; properties that make them difficult to detect and characterize in structural detail using biophysical techniques. The ability of solution NMR spectroscopy to investigate exchange between multiple protein states, to characterize transient and low-population species, and to study high molecular weight assemblies, render NMR an invaluable technique for studies of amyloid assembly. In this article we review state-of-the-art solution NMR methods for investigations of: (a) protein dynamics that lead to the formation of aggregation-prone species; (b) amyloidogenic intrinsically disordered proteins; and (c) protein-protein interactions on pathway to fibril formation. Together, these topics highlight the power and potential of NMR to provide atomic level information about the molecular mechanisms of one of the most fascinating problems in structural biology.

The structure of the PanD/PanZ protein complex reveals negative feedback regulation of pantothenate biosynthesis by coenzyme A.

Coenzyme A (CoA) is an ubiquitous and essential cofactor, synthesized from the precursor pantothenate. Vitamin biosynthetic pathways are normally tightly regulated, including the pathway from pantothenate to CoA. However, no regulation of pantothenate biosynthesis has been identified. We have recently described an additional component in the pantothenate biosynthetic pathway, PanZ, which promotes the activation of the zymogen, PanD, to form aspartate α-decarboxylase (ADC) in a CoA-dependent manner. Here we report the structure of PanZ in complex with PanD, which reveals the structural basis for the CoA dependence of this interaction and activation. In addition, we show that PanZ acts as a CoA-dependent inhibitor of ADC catalysis. This inhibitory effect can effectively regulate the biosynthetic pathway to pantothenate, and thereby also regulate CoA biosynthesis. This represents a previously unobserved mode of metabolic regulation whereby a cofactor-utilizing protein negatively regulates the biosynthesis of the same cofactor.

Deuterated detergents for structural and functional studies of membrane proteins: Properties, chemical synthesis and applications.

Detergents are amphiphilic compounds that have crucial roles in the extraction, purification and stabilization of integral membrane proteins and in experimental studies of their structure and function. One technique that is highly dependent on detergents for solubilization of membrane proteins is solution-state NMR spectroscopy, where detergent micelles often serve as the best membrane mimetic for achieving particle sizes that tumble fast enough to produce high-resolution and high-sensitivity spectra, although not necessarily the best mimetic for a biomembrane. For achieving the best quality NMR spectra, detergents with partial or complete deuteration can be used, which eliminate interfering proton signals coming from the detergent itself and also eliminate potential proton relaxation pathways and strong dipole-dipole interactions that contribute line broadening effects. Deuterated detergents have also been used to solubilize membrane proteins for other experimental techniques including small angle neutron scattering and single-crystal neutron diffraction and for studying membrane proteins immobilized on gold electrodes. This is a review of the properties, chemical synthesis and applications of detergents that are currently commercially available and/or that have been synthesized with partial or complete deuteration. Specifically, the detergents are sodium dodecyl sulphate (SDS), lauryldimethylamine-oxide (LDAO), n-octyl-ß-D-glucoside (ß-OG), n-dodecyl-ß-D-maltoside (DDM) and fos-cholines including dodecylphosphocholine (DPC). The review also considers effects of deuteration, detergent screening and guidelines for detergent selection. Although deuterated detergents are relatively expensive and not always commercially available due to challenges associated with their chemical synthesis, they will continue to play important roles in structural and functional studies of membrane proteins, especially using solution-state NMR.

Synthesis of uniformly deuterated n-dodecyl-ß-D-maltoside (d39-DDM) for solubilization of membrane proteins in TROSY NMR experiments.

This work reports the first synthesis of uniformly deuterated n-dodecyl-ß-D-maltoside (d39-DDM). DDM is a mild non-ionic detergent often used in the extraction and purification of membrane proteins and for solubilizing them in experimental studies of their structure, dynamics and binding of ligands. We required d39-DDM for solubilizing large α-helical membrane proteins in samples for [(15)N-(1)H]TROSY (transverse relaxation-optimized spectroscopy) NMR experiments to achieve the highest sensitivity and best resolved spectra possible. Our synthesis of d39-DDM used d7-D-glucose and d25-n-dodecanol to introduce deuterium labelling into both the maltoside and dodecyl moieties, respectively. Two glucose molecules, one converted to a glycosyl acceptor with a free C4 hydroxyl group and one converted to a glycosyl donor substituted at C1 with a bromine in the α-configuration, were coupled together with an α(1 → 4) glycosidic bond to give maltose, which was then coupled with n-dodecanol by its substitution of a C1 bromine in the α-configuration to give DDM. (1)H NMR spectra were used to confirm a high level of deuteration in the synthesized d39-DDM and to demonstrate its use in eliminating interfering signals from TROSY NMR spectra of a 52-kDa sugar transport protein solubilized in DDM.

Distinguishing closely related amyloid precursors using an RNA aptamer.

Although amyloid fibrils assembled in vitro commonly involve a single protein, fibrils formed in vivo can contain multiple protein sequences. The amyloidogenic protein human ß2-microglobulin (hß2m) can co-polymerize with its N-terminally truncated variant (ΔN6) in vitro to form hetero-polymeric fibrils that differ from their homo-polymeric counterparts. Discrimination between the different assembly precursors, for example by binding of a biomolecule to one species in a mixture of conformers, offers an opportunity to alter the course of co-assembly and the properties of the fibrils formed. Here, using hß2m and its amyloidogenic counterpart, ΔΝ6, we describe selection of a 2'F-modified RNA aptamer able to distinguish between these very similar proteins. SELEX with a N30 RNA pool yielded an aptamer (B6) that binds hß2m with an EC50 of ∼200 nM. NMR spectroscopy was used to assign the (1)H-(15)N HSQC spectrum of the B6-hß2m complex, revealing that the aptamer binds to the face of hß2m containing the A, B, E, and D ß-strands. In contrast, binding of B6 to ΔN6 is weak and less specific. Kinetic analysis of the effect of B6 on co-polymerization of hß2m and ΔN6 revealed that the aptamer alters the kinetics of co-polymerization of the two proteins. The results reveal the potential of RNA aptamers as tools for elucidating the mechanisms of co-assembly in amyloid formation and as reagents able to discriminate between very similar protein conformers with different amyloid propensity.

Visualization of transient protein-protein interactions that promote or inhibit amyloid assembly.

In the early stages of amyloid formation, heterogeneous populations of oligomeric species are generated, the affinity, specificity, and nature of which may promote, inhibit, or define the course of assembly. Despite the importance of the intermolecular interactions that initiate amyloid assembly, our understanding of these events remains poor. Here, using amyloidogenic and nonamyloidogenic variants of ß2-microglobulin, we identify the interactions that inhibit or promote fibril formation in atomic detail. The results reveal that different outcomes of assembly result from biomolecular interactions involving similar surfaces. Specifically, inhibition occurs via rigid body docking of monomers in a head-to-head orientation to form kinetically trapped dimers. By contrast, the promotion of fibrillation involves relatively weak protein association in a similar orientation, which results in conformational changes in the initially nonfibrillogenic partner. The results highlight the complexity of interactions early in amyloid assembly and reveal atomic-level information about species barriers in amyloid formation.