Researchers unravel information about a protein causing progressive diseases
The C-D loop in transthyretin is tight in the stable T119M mutant (protective against amyloidosis). However, the same loop at the mutation site of the S52P mutant (associated with the most aggressive form of transthyretin amyloidosis) is loosened, causing degradation of the protein and misfolding into damaging amyloid fibrillary deposits.
Advanced analysis of a blood hormone transporter protein using neutron diffraction, mass spectrometry, molecular modelling, and X-ray diffraction, is revealing the mechanisms underlying this invariably fatal disease and how it may be prevented.
Transthyretin amyloidosis is a progressive condition in which abnormally folded forms of an important hormone transporter protein accumulate as amyloid fibrils/plaques in various parts of the body – resulting in the condition known as familial amyloid polyneuropathy (FAP). The most affected tissues include the peripheral nervous system (sensory perception - pain, touch, heat, sound), the autonomic nervous system (involuntary functions such as breathing, heart rate, digestion). Other regions such as the central nervous system, the heart, the kidneys and the gastrointestinal tract may also be afflicted.
In this study, the scientists have investigated normal human transthyretin alongside two variants of the protein (mutants that are found in different parts of the population). One of these (the T119M or ‘stable’ mutant) imparts remarkable stability to transthyretin and is strongly protective against the formation of amyloid fibrils. However the other (the S52P or ‘unstable’ mutant) results in a very aggressive form of hereditary amyloidosis.
All of the transthyretin mutants were studied using neutron and X-ray crystallography, along with mass spectrometry and computer modelling. Crucially, the project was able to exploit the technologies available within the Deuteration Laboratory (D-Lab) platform of ILL’s Life Sciences Group [2]. These investigations have resulted in the proposal of a molecular mechanism by which transthyretin forms amyloid fibrils through a parallel equilibrium of partially unfolded protein forms.
Transthyretin has a complex hierarchical structure of four subunits (monomers). The results suggest that the disease progression arises from instability in a particular part of the protein called the C-D loop. This instability renders the whole protein more susceptible to degradation effects, enhancing the rate of amyloid fibril formation. Furthermore, the study suggests that the binding of small molecule drugs to transthyretin stabilises the folded state of transthyretin in the same way as happens in the highly stable protein mutants.
Molecular dynamics simulations show the nature of the destablisation pathway from the fully folded, functional TTR towards amyloid fibrilisation.
A new neutron method
Additionally, this study has emphasised the potential exploitability of a new neutron approach that can be used to provide important information on the dynamics and stability of particular regions of a protein. Neutron crystallography is usually carried out with all of the ‘normal’ water (H2O) replaced by ‘heavy’ water (D2O) – this is done for technical reasons and helps to optimise the quality of the data recorded. However, controlled ‘back-exchange’ of D2O by H2O can be carried out [3]. This solvent exchange occurs more readily in regions of the protein that have greater flexibility and movement - regions that are more susceptible to denaturation, misfolding, and amyloid fibril formation. This type of approach has been successfully used in NMR and mass spectrometry work but until now has never been seriously applied in neutron crystallographic studies.
In the case of transthyretin, the back-exchange data recorded using LADI’s neutron diffractometer from fully deuterated transthyretin crystals demonstrate very clear differences in the patterns of stability amongst the transthyretin mutants – with the S52P mutant showing further evidence in support of a highly unstable fold, consistent with the computer modelling work. Intriguingly, recent research by colleagues at University College London (UCL) has implicated plasminogen – a precursor of plasmin (which breaks up blood clots) – in transthyretin degradation and amyloidosis [4]. This observation may be related to increased accessibility in or near the C-D loop which allows the enzyme to cleave the protein at this location.
Reference: A. Yee et al., Nature Communications 2019 : 10 : 925. doi : 10.1038/s41467-019-08609-z
Contact: Dr Trevor Forsyth
Research team: Alycia Yee (Keele University (UK)), Matteo Aldeghi (MPI, Göttingen, Germany), Matthew Blakeley, Martine Moulin, Michael Haertlein and Trevor Forsyth (ILL)
Instrument/facilities used:Deuteration Laboratory (ILL Life Sciences Group), LADI-III (Large Scale Structures Group), PSB (Partnership for Structural Biology).