ABSTRACT
Enzymes facilitating the transfer of phosphate groups constitute the most extensive protein families across all kingdoms of life. They make up approximately 10% of the proteins found in the human genome. Understanding the mechanisms by which enzymes catalyze these reactions is essential in characterizing the processes they regulate. Metal fluorides can be used as multifunctional tools to study these enzymes. These ionic species bear the same charge as phosphate and the transferring phosphoryl group and, in addition, allow the enzyme to be trapped in catalytically important states with spectroscopically sensitive atoms interacting directly with active site residues. The ionic nature of these phosphate surrogates also allows their removal and replacement with other analogs. Here, we describe the best practices to obtain these complexes, their use in NMR, X-ray crystallography, cryo-EM, and SAXS and describe a new metal fluoride, scandium tetrafluoride, which has significant anomalous signal using soft X-rays.
ABSTRACT
The advent of serial crystallography has rejuvenated and popularized room-temperature X-ray crystal structure determination. Structures determined at physiological temperature reveal protein flexibility and dynamics. In addition, challenging samples (e.g. large complexes, membrane proteins and viruses) form fragile crystals that are often difficult to harvest for cryo-crystallography. Moreover, a typical serial crystallography experiment requires a large number of microcrystals, mainly achievable through batch crystallization. Many medically relevant samples are expressed in mammalian cell lines, producing a meager quantity of protein that is incompatible with batch crystallization. This can limit the scope of serial crystallography approaches. Direct in situ data collection from a 96-well crystallization plate enables not only the identification of the best diffracting crystallization condition but also the possibility for structure determination under ambient conditions. Here, we describe an in situ serial crystallography (iSX) approach, facilitating direct measurement from crystallization plates mounted on a rapidly exchangeable universal plate holder deployed at a microfocus beamline, ID23-2, at the European Synchrotron Radiation Facility. We applied our iSX approach on a challenging project, autotaxin, a therapeutic target expressed in a stable human cell line, to determine the structure in the lowest-symmetry P1 space group at 3.0â Å resolution. Our in situ data collection strategy provided a complete dataset for structure determination while screening various crystallization conditions. Our data analysis reveals that the iSX approach is highly efficient at a microfocus beamline, improving throughput and demonstrating how crystallization plates can be routinely used as an alternative method of presenting samples for serial crystallography experiments at synchrotrons.
ABSTRACT
Fragment-based drug design using X-ray crystallography is a powerful technique to enable the development of new lead compounds, or probe molecules, against biological targets. This study addresses the need to determine fragment binding orientations for low-occupancy fragments with incomplete electron density, an essential step before further development of the molecule. Halogen atoms play multiple roles in drug discovery due to their unique combination of electronegativity, steric effects and hydrophobic properties. Fragments incorporating halogen atoms serve as promising starting points in hit-to-lead development as they often establish halogen bonds with target proteins, potentially enhancing binding affinity and selectivity, as well as counteracting drug resistance. Here, the aim was to unambiguously identify the binding orientations of fragment hits for SARS-CoV-2 nonstructural protein 1 (nsp1) which contain a combination of sulfur and/or chlorine, bromine and iodine substituents. The binding orientations of carefully selected nsp1 analogue hits were focused on by employing their anomalous scattering combined with Pan-Dataset Density Analysis (PanDDA). Anomalous difference Fourier maps derived from the diffraction data collected at both standard and long-wavelength X-rays were compared. The discrepancies observed in the maps of iodine-containing fragments collected at different energies were attributed to site-specific radiation-damage stemming from the strong X-ray absorption of I atoms, which is likely to cause cleavage of the C-I bond. A reliable and effective data-collection strategy to unambiguously determine the binding orientations of low-occupancy fragments containing sulfur and/or halogen atoms while mitigating radiation damage is presented.
Subject(s)
Halogens , SARS-CoV-2 , Sulfur , Halogens/chemistry , Crystallography, X-Ray/methods , Sulfur/chemistry , SARS-CoV-2/chemistry , Viral Nonstructural Proteins/chemistry , Humans , Electrons , Models, Molecular , Drug Design , Protein Binding , Binding Sites , COVID-19ABSTRACT
The Mitochondrial Complex I Assembly (MCIA) complex is essential for the biogenesis of respiratory Complex I (CI), the first enzyme in the respiratory chain, which has been linked to Alzheimer's disease (AD) pathogenesis. However, how MCIA facilitates CI assembly, and how it is linked with AD pathogenesis, is poorly understood. Here we report the structural basis of the complex formation between the MCIA subunits ECSIT and ACAD9. ECSIT binding induces a major conformational change in the FAD-binding loop of ACAD9, releasing the FAD cofactor and converting ACAD9 from a fatty acid ß-oxidation (FAO) enzyme to a CI assembly factor. We provide evidence that ECSIT phosphorylation downregulates its association with ACAD9 and is reduced in neuronal cells upon exposure to amyloid-ß (Aß) oligomers. These findings advance our understanding of the MCIA complex assembly and suggest a possible role for ECSIT in the reprogramming of bioenergetic pathways linked to Aß toxicity, a hallmark of AD.
Subject(s)
Alzheimer Disease , Electron Transport Complex I , Humans , Oxidation-Reduction , Electron Transport Complex I/metabolism , Energy Metabolism , Alzheimer Disease/metabolism , Amyloid beta-Peptides/metabolismABSTRACT
The mitogen-activated protein kinase (MAPK) p38α is a central component of signaling in inflammation and the immune response and is, therefore, an important drug target. Little is known about the molecular mechanism of its activation by double phosphorylation from MAPK kinases (MAP2Ks), because of the challenge of trapping a transient and dynamic heterokinase complex. We applied a multidisciplinary approach to generate a structural model of p38α in complex with its MAP2K, MKK6, and to understand the activation mechanism. Integrating cryo-electron microscopy with molecular dynamics simulations, hydrogen-deuterium exchange mass spectrometry, and experiments in cells, we demonstrate a dynamic, multistep phosphorylation mechanism, identify catalytically relevant interactions, and show that MAP2K-disordered amino termini determine pathway specificity. Our work captures a fundamental step of cell signaling: a kinase phosphorylating its downstream target kinase.
Subject(s)
MAP Kinase Kinase 2 , MAP Kinase Kinase 6 , Mitogen-Activated Protein Kinase 14 , Cryoelectron Microscopy , Enzyme Activation , MAP Kinase Kinase 2/chemistry , MAP Kinase Kinase 6/chemistry , Mitogen-Activated Protein Kinase 14/chemistry , Phosphorylation , Substrate Specificity , Protein ConformationABSTRACT
The identification of multiple simultaneous orientations of small molecule inhibitors binding to a protein target is a common challenge. It has recently been reported that the conformational heterogeneity of ligands is widely underreported in the Protein Data Bank, which is likely to impede optimal exploitation to improve affinity of these ligands. Significantly less is even known about multiple binding orientations for fragments (<300 Da), although this information would be essential for subsequent fragment optimisation using growing, linking or merging and rational structure-based design. Here, we use recently reported fragment hits for the SARS-CoV-2 non-structural protein 1 (nsp1) N-terminal domain to propose a general procedure for unambiguously identifying binding orientations of 2-dimensional fragments containing either sulphur or chloro substituents within the wavelength range of most tunable beamlines. By measuring datasets at two energies, using a tunable beamline operating in vacuum and optimised for data collection at very low X-ray energies, we show that the anomalous signal can be used to identify multiple orientations in small fragments containing sulphur and/or chloro substituents or to verify recently reported conformations. Although in this specific case we identified the positions of sulphur and chlorine in fragments bound to their protein target, we are confident that this work can be further expanded to additional atoms or ions which often occur in fragments. Finally, our improvements in the understanding of binding orientations will also serve to improve the rational optimisation of SARS-CoV-2 nsp1 fragment hits.
Subject(s)
COVID-19 , SARS-CoV-2 , Humans , Electrons , Ligands , SynchrotronsABSTRACT
The polymorphism of human insulin upon pH variation was characterized via X-ray powder diffraction, employing a crystallization protocol previously established for co-crystallization with phenolic derivatives. Two distinct rhombohedral (R3) polymorphs and one cubic (I213) polymorph were identified with increasing pH, corresponding to the T6, T3R3f and T2 conformations of insulin, respectively. The structure of the cubic T2 polymorph was determined via multi-profile stereochemically restrained Rietveld refinement at 2.7â Å resolution. This constitutes the first cubic insulin structure to be determined from crystals grown in the presence of zinc ions, although no zinc binding was observed. The differences of the polycrystalline variant from other cubic insulin structures, as well as the nature of the pH-driven phase transitions, are discussed in detail.
Subject(s)
Insulin, Regular, Human , Insulin , Humans , Insulin/chemistry , X-Ray Diffraction , Phenols , CrystallizationABSTRACT
Programmed death-ligand 1 (PD-L1) is a key immune regulatory protein that interacts with programmed cell death protein 1 (PD-1), leading to T-cell suppression. Whilst this interaction is key in self-tolerance, cancer cells evade the immune system by overexpressing PD-L1. Inhibition of the PD-1/PD-L1 pathway with standard monoclonal antibodies has proven a highly effective cancer treatment; however, single domain antibodies (VHH) may offer numerous potential benefits. Here, we report the identification and characterization of a diverse panel of 16 novel VHHs specific to PD-L1. The panel of VHHs demonstrate affinities of 0.7 nM to 5.1 µM and were able to completely inhibit PD-1 binding to PD-L1. The binding site for each VHH on PD-L1 was determined using NMR chemical shift perturbation mapping and revealed a common binding surface encompassing the PD-1-binding site. Additionally, we solved crystal structures of two representative VHHs in complex with PD-L1, which revealed unique binding modes. Similar NMR experiments were used to identify the binding site of CD80 on PD-L1, which is another immune response regulatory element and interacts with PD-L1 localized on the same cell surface. CD80 and PD-1 were revealed to share a highly overlapping binding site on PD-L1, with the panel of VHHs identified expected to inhibit CD80 binding. Comparison of the CD80 and PD-1 binding sites on PD-L1 enabled the identification of a potential antibody binding region able to confer specificity for the inhibition of PD-1 binding only, which may offer therapeutic benefits to counteract cancer cell evasion of the immune system.
Subject(s)
Antibodies , B7-1 Antigen , B7-H1 Antigen , Programmed Cell Death 1 Receptor , Humans , B7-1 Antigen/metabolism , B7-H1 Antigen/genetics , B7-H1 Antigen/metabolism , Neoplasms/therapy , Programmed Cell Death 1 Receptor/metabolism , Protein Binding , Binding Sites , Crystallography , Antibodies/chemistry , Antibodies/metabolismABSTRACT
The regular reappearance of coronavirus (CoV) outbreaks over the past 20 years has caused significant health consequences and financial burdens worldwide. The most recent and still ongoing novel CoV pandemic, caused by Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) has brought a range of devastating consequences. Due to the exceptionally fast development of vaccines, the mortality rate of the virus has been curbed to a significant extent. However, the limitations of vaccination efficiency and applicability, coupled with the still high infection rate, emphasise the urgent need for discovering safe and effective antivirals against SARS-CoV-2 by suppressing its replication or attenuating its virulence. Non-structural protein 1 (nsp1), a unique viral and conserved leader protein, is a crucial virulence factor for causing host mRNA degradation, suppressing interferon (IFN) expression and host antiviral signalling pathways. In view of the essential role of nsp1 in the CoV life cycle, it is regarded as an exploitable target for antiviral drug discovery. Here, we report a variety of fragment hits against the N-terminal domain of SARS-CoV-2 nsp1 identified by fragment-based screening via X-ray crystallography. We also determined the structure of nsp1 at atomic resolution (0.99 Å). Binding affinities of hits against nsp1 and potential stabilisation were determined by orthogonal biophysical assays such as microscale thermophoresis and thermal shift assays. We identified two ligand-binding sites on nsp1, one deep and one shallow pocket, which are not conserved between the three medically relevant SARS, SARS-CoV-2 and MERS coronaviruses. Our study provides an excellent starting point for the development of more potent nsp1-targeting inhibitors and functional studies on SARS-CoV-2 nsp1.
Subject(s)
COVID-19 , SARS-CoV-2 , Humans , Viral Nonstructural Proteins/metabolism , Ligands , X-Rays , Binding Sites , Antiviral Agents/pharmacology , Interferons , Virulence FactorsABSTRACT
The Apicomplexa comprise a large phylum of single-celled, obligate intracellular protozoa that include Toxoplasma gondii, Plasmodium, and Cryptosporidium spp., which infect humans and animals and cause severe parasitic diseases. Available therapeutics against these diseases are limited by suboptimal efficacy and frequent side effects, as well as the emergence and spread of resistance. We use a drug repurposing strategy and identify altiratinib, a compound originally developed to treat glioblastoma, as a promising drug candidate with broad spectrum activity against apicomplexans. Altiratinib is parasiticidal and blocks the development of intracellular zoites in the nanomolar range and with a high selectivity index when used against T. gondii. We have identified TgPRP4K of T. gondii as the primary target of altiratinib using genetic target deconvolution, which highlighted key residues within the kinase catalytic site that conferred drug resistance when mutated. We have further elucidated the molecular basis of the inhibitory mechanism and species selectivity of altiratinib for TgPRP4K and for its Plasmodium falciparum counterpart, PfCLK3. Our data identified structural features critical for binding of the other PfCLK3 inhibitor, TCMDC-135051. Consistent with the splicing control activity of this kinase family, we have shown that altiratinib can cause global disruption of splicing, primarily through intron retention in both T. gondii and P. falciparum. Thus, our data establish parasitic PRP4K/CLK3 as a potential pan-apicomplexan target whose repertoire of inhibitors can be expanded by the addition of altiratinib.
Subject(s)
Cryptosporidiosis , Cryptosporidium , Malaria, Falciparum , Toxoplasma , Angiogenesis Inhibitors/therapeutic use , Animals , Humans , Malaria, Falciparum/drug therapy , Plasmodium falciparum , Protein Kinase Inhibitors/pharmacology , Spliceosomes , Toxoplasma/geneticsABSTRACT
Non-ribosomal peptide synthetases (NRPS) are multi-modular/domain enzymes that catalyze the synthesis of bioactive peptides. A crucial step in the process is peptide elongation accomplished by the condensation (C) domain with the aid of a peptidyl carrier or thiolation (T) domain. Here, we examined condensation reaction carried out by NRPS AmbB involved in biosynthesis of L-2-amino-4-methoxy-trans-3-butenoic acid (AMB) in P. aeruginosa. We determined crystal structures of the truncated T-C bidomain of AmbB in three forms, the apo enzyme with disordered T domain, the holo form with serine linked phosphopantetheine (Ppant) and a holo form with substrate (L-alanine) loaded onto Ppant. The two holo forms feature the T domain in a substrate-donation conformation. Mutagenesis combined with functional assays identified residues essential for the attachment of Ppant, anchoring the Ppant-L-Ala in the donor catalytic channel and the role of the conserved His953 in condensation activity. Altogether, these results provide structural insights into the condensation reaction at the donor site with a substrate-bound C domain of AmbB and lay the foundation for understanding the molecular mechanism of condensation which is crucial for AMB synthesis.
Subject(s)
Peptide Synthases , Catalytic Domain , Peptide Synthases/metabolism , Protein Domains , Protein Structure, TertiaryABSTRACT
Correct 3'end processing of mRNAs is one of the regulatory cornerstones of gene expression. In a parasite that must adapt to the regulatory requirements of its multi-host life style, there is a need to adopt additional means to partition the distinct transcriptional signatures of the closely and tandemly arranged stage-specific genes. In this study, we report our findings in T. gondii of an m6A-dependent 3'end polyadenylation serving as a transcriptional barrier at these loci. We identify the core polyadenylation complex within T. gondii and establish CPSF4 as a reader for m6A-modified mRNAs, via a YTH domain within its C-terminus, a feature which is shared with plants. We bring evidence of the specificity of this interaction both biochemically, and by determining the crystal structure at high resolution of the T. gondii CPSF4-YTH in complex with an m6A-modified RNA. We show that the loss of m6A, both at the level of its deposition or its recognition is associated with an increase in aberrantly elongated chimeric mRNAs emanating from impaired transcriptional termination, a phenotype previously noticed in the plant model Arabidopsis thaliana. Nanopore direct RNA sequencing shows the occurrence of transcriptional read-through breaching into downstream repressed stage-specific genes, in the absence of either CPSF4 or the m6A RNA methylase components in both T. gondii and A. thaliana. Taken together, our results shed light on an essential regulatory mechanism coupling the pathways of m6A metabolism directly to the cleavage and polyadenylation processes, one that interestingly seem to serve, in both T. gondii and A. thaliana, as a guardian against aberrant transcriptional read-throughs.
Subject(s)
Genes, Developmental , Membrane Glycoproteins/metabolism , Nerve Tissue Proteins/metabolism , Polyadenylation , Toxoplasma/metabolism , Transcriptome , Arabidopsis/genetics , Binding Sites , Cleavage And Polyadenylation Specificity Factor/metabolism , Gene Expression Regulation , Humans , Membrane Glycoproteins/chemistry , Methyltransferases/metabolism , Models, Molecular , Nerve Tissue Proteins/chemistry , RNA Splicing Factors/chemistry , RNA, Messenger/metabolism , Reading , Sequence Analysis, RNA , Zinc FingersABSTRACT
Passive immunization using monoclonal antibodies will play a vital role in the fight against COVID-19. The recent emergence of viral variants with reduced sensitivity to some current antibodies and vaccines highlights the importance of broad cross-reactivity. This study describes deep-mining of the antibody repertoires of hospitalized COVID-19 patients using phage display technology and B cell receptor (BCR) repertoire sequencing to isolate neutralizing antibodies and gain insights into the early antibody response. This comprehensive discovery approach has yielded a panel of potent neutralizing antibodies which bind distinct viral epitopes including epitopes conserved in SARS-CoV-1. Structural determination of a non-ACE2 receptor blocking antibody reveals a previously undescribed binding epitope, which is unlikely to be affected by the mutations in any of the recently reported major viral variants including B.1.1.7 (from the UK), B.1.351 (from South Africa) and B.1.1.28 (from Brazil). Finally, by combining sequences of the RBD binding and neutralizing antibodies with the B cell receptor repertoire sequencing, we also describe a highly convergent early antibody response. Similar IgM-derived sequences occur within this study group and also within patient responses described by multiple independent studies published previously.
Subject(s)
Antibodies, Monoclonal/therapeutic use , Antibodies, Neutralizing/therapeutic use , COVID-19/prevention & control , COVID-19/therapy , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/immunology , Cell Surface Display Techniques/methods , Data Mining/methods , Epitopes/immunology , Humans , Immunization, Passive/methods , COVID-19 SerotherapyABSTRACT
FAH domain containing protein 1 (FAHD1) is a mammalian mitochondrial protein, displaying bifunctionality as acylpyruvate hydrolase (ApH) and oxaloacetate decarboxylase (ODx) activity. We report the crystal structure of mouse FAHD1 and structural mapping of the active site of mouse FAHD1. Despite high structural similarity with human FAHD1, a rabbit monoclonal antibody (RabMab) could be produced that is able to recognize mouse FAHD1, but not the human form, whereas a polyclonal antibody recognized both proteins. Epitope mapping in combination with our deposited crystal structures revealed that the epitope overlaps with a reported SIRT3 deacetylation site in mouse FAHD1.
Subject(s)
Hydrolases/genetics , Acetoacetates/metabolism , Animals , Carboxy-Lyases/metabolism , Catalytic Domain , Crystallography, X-Ray , Epitope Mapping/methods , Humans , Hydrolases/chemistry , Hydrolases/metabolism , Mice , Mitochondria/metabolism , Mitochondrial Proteins/metabolism , Structure-Activity RelationshipABSTRACT
Afamin, which is a human blood plasma glycoprotein, a putative multifunctional transporter of hydrophobic molecules and a marker for metabolic syndrome, poses multiple challenges for crystallographic structure determination, both practically and in analysis of the models. Several hundred crystals were analysed, and an unusual variability in cell volume and difficulty in solving the structure despite an â¼34% sequence identity with nonglycosylated human serum albumin indicated that the molecule exhibits variable and context-sensitive packing, despite the simplified glycosylation in insect cell-expressed recombinant afamin. Controlled dehydration of the crystals was able to stabilize the orthorhombic crystal form, reducing the number of molecules in the asymmetric unit from the monoclinic form and changing the conformational state of the protein. An iterative strategy using fully automatic experiments available on MASSIF-1 was used to quickly determine the optimal protocol to achieve the phase transition, which should be readily applicable to many types of sample. The study also highlights the drawback of using a single crystallographic structure model for computational modelling purposes given that the conformational state of the binding sites and the electron density in the binding site, which is likely to result from PEGs, greatly varies between models. This also holds for the analysis of nonspecific low-affinity ligands, where often a variety of fragments with similar uncertainty can be modelled, inviting interpretative bias. As a promiscuous transporter, afamin also seems to bind gadoteridol, a magnetic resonance imaging contrast compound, in at least two sites. One pair of gadoteridol molecules is located near the human albumin Sudlow site, and a second gadoteridol molecule is located at an intermolecular site in proximity to domain IA. The data from the co-crystals support modern metrics of data quality in the context of the information that can be gleaned from data sets that would be abandoned on classical measures.
Subject(s)
Carrier Proteins/chemistry , Crystallization/methods , Desiccation/methods , Glycoproteins/chemistry , Serum Albumin, Human/chemistry , Binding Sites , Gadolinium/chemistry , Heterocyclic Compounds/chemistry , Hydrophobic and Hydrophilic Interactions , Ligands , Organometallic Compounds/chemistry , Protein ConformationABSTRACT
The fully automatic processing of crystals of macromolecules has presented a unique opportunity to gather information on the samples that is not usually recorded. This has proved invaluable in improving sample-location, characterization and data-collection algorithms. After operating for four years, MASSIF-1 has now processed over 56â 000 samples, gathering information at each stage, from the volume of the crystal to the unit-cell dimensions, the space group, the quality of the data collected and the reasoning behind the decisions made in data collection. This provides an unprecedented opportunity to analyse these data together, providing a detailed landscape of macromolecular crystals, intimate details of their contents and, importantly, how the two are related. The data show that mosaic spread is unrelated to the size or shape of crystals and demonstrate experimentally that diffraction intensities scale in proportion to crystal volume and molecular weight. It is also shown that crystal volume scales inversely with molecular weight. The results set the scene for the development of X-ray crystallography in a changing environment for structural biology.
ABSTRACT
High-brilliance X-ray beams coupled with automation have led to the use of synchrotron-based macromolecular X-ray crystallography (MX) beamlines for even the most challenging projects in structural biology. However, most facilities still require the presence of a scientist on site to perform the experiments. A new generation of automated beamlines dedicated to the fully automatic characterization of, and data collection from, crystals of biological macromolecules has recently been developed. These beamlines represent a new tool for structural biologists to screen the results of initial crystallization trials and/or the collection of large numbers of diffraction data sets, without users having to control the beamline themselves. Here we show how to set up an experiment for automatic screening and data collection, how an experiment is performed at the beamline, how the resulting data sets are processed, and how, when possible, the crystal structure of the biological macromolecule is solved.
Subject(s)
Crystallography, X-Ray , Macromolecular Substances/chemistry , Crystallography, X-Ray/instrumentation , Data Collection , SynchrotronsABSTRACT
MXCuBE2 is the second-generation evolution of the MXCuBE beamline control software, initially developed and used at ESRF - the European Synchrotron. MXCuBE2 extends, in an intuitive graphical user interface (GUI), the functionalities and data collection methods available to users while keeping all previously available features and allowing for the straightforward incorporation of ongoing and future developments. MXCuBE2 introduces an extended abstraction layer that allows easy interfacing of any kind of macromolecular crystallography (MX) hardware component, whether this is a diffractometer, sample changer, detector or optical element. MXCuBE2 also works in strong synergy with the ISPyB Laboratory Information Management System, accessing the list of samples available for a particular experimental session and associating, either from instructions contained in ISPyB or from user input via the MXCuBE2 GUI, different data collection types to them. The development of MXCuBE2 forms the core of a fruitful collaboration which brings together several European synchrotrons and a software development factory and, as such, defines a new paradigm for the development of beamline control platforms for the European MX user community.
ABSTRACT
Human transthyretin (TTR) is implicated in several fatal forms of amyloidosis. Many mutations of TTR have been identified; most of these are pathogenic, but some offer protective effects. The molecular basis underlying the vastly different fibrillation behaviours of these TTR mutants is poorly understood. Here, on the basis of neutron crystallography, native mass spectrometry and modelling studies, we propose a mechanism whereby TTR can form amyloid fibrils via a parallel equilibrium of partially unfolded species that proceeds in favour of the amyloidogenic forms of TTR. It is suggested that unfolding events within the TTR monomer originate at the C-D loop of the protein, and that destabilising mutations in this region enhance the rate of TTR fibrillation. Furthermore, it is proposed that the binding of small molecule drugs to TTR stabilises non-amyloidogenic states of TTR in a manner similar to that occurring for the protective mutants of the protein.