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Structure and Dynamics of Low-Complexity Regions in Proteins: The Huntingtin Case

Periodic Reporting for period 2 - chemREPEAT (Structure and Dynamics of Low-Complexity Regions in Proteins: The Huntingtin Case)

Reporting period: 2017-03-01 to 2018-08-31

The main aim of chemREPEAT is the structural characterization of protein huntingtin (htt), the causative agent of Huntington’s Disease (HD), and to understand the structural bases of this pathology. The N-terminal region of Htt, the so-called exon1, contains a homorepeat (HR) region that contains a large number of consecutive glutamine residues. Individuals with more than 35 consecutive glutamines suffer this deadly neurodegenerative pathology. The structural characterization of htt represents an enormous challenge due to its inherent flexibility, which precludes the use of X-ray crystallography, and its repetitive nature that hampers the application of Nuclear Magnetic Resonance (NMR). Our developments aim at surpassing present limitation and shedding light to fundamental aspects of this disease. Our efforts in this period have been centred in developing strategies that will enable us to tackle the challenge and to reach structural/dynamic models of the Htt. by combining experimental and computational approaches.
We can divide our work in two main aspects that are directly related with the experimental (points 1-4) and computational (points 5 and 6) parts that will merge in the future to derive an atomistic picture of the structural bases of the pathological threshold in HD.

1. Residue specific labelling within poly-Q regions: The proof-of-concept
Isotopic labelling, which is necessary to apply NMR, yields htt samples in which the Glutamine peaks appear in a narrow region of the spectra precluding the traditional frequency assignment that is necessary for subsequent structural studies. To disentangle this complexity we have designed a strategy enabling isotopic labelling in a glutamine-specific manner. In this way, NMR spectrum is reduced to a single peak that probes the structure and dynamics of an individual glutamine within the Poly-Q homorepeat. The strategy consists in the combination of cell-free protein expression with tRNA non-sense suppression. In this period we have developed both tools and, by combining them, we proved the validity of the concept. Briefly:
(i) We have adapted a published protocol to produce efficient E.coli lysates and methods to optimize cell-free protein synthesis.
(ii) We can produce and purify properly folded tRNA in large quantities.
(iii) We overexpress and purify an active engineered yeast glutaminyl tRNA synthetase enabling an efficient loading of the glutamine amino acid to the tRNA.
(iv) We have optimized an efficient way to introduce loaded tRNA into cell-free reactions to obtain the residue-specific isotopically labelled htt.
(v) We have adapted the protein construct (fused to a Green-Fluorescent Protein and a His-tag) to monitor and efficiently purify the protein for subsequent NMR studies.
(vi) We have adapted NMR pulse sequences to record in moderate time 15N-H and 13C-H NMR spectra.
These developments are described in a recent publication in which 5 glutamines of a non-pathological version of Htt exon1 (H16) were studied [Urbanek et al. Angewandte Chemie, 2018]. Moreover, parts of this work have been presented in national and international conferences (see Below)

2. Structural investigation of the non-pathological version of Htt exon1 H16.
Using the above described methodology we have addressed the structural and dynamic characterization of a non-pathological version of Htt exon1 containing 16 consecutive glutamines (H16). We scanned the 22 glutamines of the construct (16 from the homorepeat and 6 from the flanking regions) by producing residue-specific labelled samples for which we measured 15N-H and 13C-H NMR spectra to obtain precise backbone and side-chain chemical shifts. Moreover, using standard 3D NMR experiments we assigned the rest of the residues of H16 (with exception of the prolines). The primary analysis of the data indicates the presence of an alpha-helix encompassing the N-terminal region of H16, the so-called N17, and the initial part of the poly-Q homorepeat. The helical propensity smoothly decreases along the homorepeat to become fully disordered in the last residues of the homorepeat. At present we are performing an ensemble model driven by the experimental chemical shifts. In the following weeks we will proceed to write and submit a manuscript describing these results.
This work has been presented already in a local conference, and it will be presented in two others in the following months (see below).

3. Application of pathological versions of Htt.
Unveiling the structural bases of the pathological threshold of HD depends on the capacity to perform the above-mentioned experiments to a pathological version of Htt. This is not straightforward as long Poly-Q tracts are prone to aggregation. We have optimized the protein production and purification of a Htt construct containing 46 consecutive glutamines fused to the GFP and a his-tag (H46). These preliminary results have been reported in the previously mentioned article [Urbanek et al. Angewandte Chemie 2018]. NMR experi
LCRs have remained out of the reach of structural biology methods. The methodologies that we are developing are surpassing this barrier and will enable a detailed understanding of the structural bases of biological and pathological phenomena involving LCRs. Huntingtin, the subject of our study, is arguably the most notable example of the connection between LCRs and pathology. These are the main achievements and how we will exploit them until the end of the project:

- Methods enabling the site-specific isotopic labelling will provide for the first time the clues of the structural/dynamic bases of Huntington’s disease. As initially planned, we will do this by systematically studying Htt versions below (already done), above (H46) and in the pathological threshold (H35).

- The developments performed to site-specifically label glutamine and proline have prompted us to span the panel of natural amino acids amenable to this kind of incorporation. We expect these developments that will be performed in the second part of the project to be a breakthrough in structural biology as multiple biologically relevant systems will be addressable for structural biologists.

- Along the project we have developed efficient cell-free methods for the in vitro synthesis of Htt variants. Now we want to exploit this achievement to produce Htt versions with different deuteration schemes (Gln, Pro, Gln+Pro…) to obtain domain specific structural information using Small-Angle Neutron Scattering (SANS). I have contacted experts at the Institute Laue-Langevin (ILL-Grenoble) to co-supervise a PhD student starting in 2019.

One of the main problems in the characterization of IDPs is the need for extensive experimental data to derive accurate structural models, and the inability of purely theoretical methods to achieve this aim. We have developed an approach based in a coil library that overcomes previous limitations and enables the construction of accurate ensemble models of IDPs embedding partially structured regions. In the future we will use this tool for several objectives:

- A server will be built and made available to all the community for the generation of accurate ensembles of IDPs from protein sequences.

- The ensembles built with this approach will be used to structurally characterize Htt by integrating the experimental data that will be produced along the project.

- Structural models built with this approach will be used as starting points to build conformation transition pathways with the robotics-inspired algorithm (multi-TRRT) that we are developing with Juan Cortés (LAAS-Toulouse).