Structure Zoom, Zooming in on protein functional sites with atomic resolution - an integrated chemistry approach for structural biology

The project “Structure Zoom” has focused on developing and combining existing chemical tools to access site-specifically isotope-labelled protein samples that allow “zooming” into their structure to visualize local structure changes and linking them to functional consequences. These tools have been applied to a variety of medically relevant protein targets (Tau, α-actinin-1, FATZ-1, HMGN-1, CD44) to study these by a combination of structural biology techniques (mainly NMR) between March 2018 and December 2022. Three of these target proteins (Tau, FATZ-1 and HMGN-1) are inherently difficult to analyse structurally as the belong to the class in intrinsically disordered proteins (IPDs).

A first highlight was achieved by establishing a robust semi-synthesis strategy for full-length Tau protein, a causative protein in the development of Alzheimer’s disease, which has now been used to generate more than 10 different segmentally isotope-labelled and posttranslationally modified Tau variants. These modifications are currently investigated with respect to their impact on disease states. Another impressive showcase in our hands for the power of segmental and amino acid-specific labelling is α-actinin-1, a large protein complex that plays a major role in controlling muscle activity. Here we could observe the effect of Ca2+-binding by shifting signals in NMR thanks to a highly flexible isotope labelling scheme, which will help to understand organization and activation of muscle fibres. Our work on FATZ-1, another muscle organizing protein, is based on a combination of structural biology techniques – NMR, SAXS, and X-ray crystallography - combined with biophysical and biochemical analysis to show that FATZ-1 forms a complex with α-actinin-2, and proposes a molecular mechanism for this interaction. We also found evidence for the ability of FATZ-1 to phase-separate and form biomolecular condensates with α-actinin-2, leading to a tantalizing hypothesis that liquid-liquid phase separation is involved in the initial stages of muscle fibre formation. Segmentally labelled high-mobility group nucleosome-binding protein HMGN1 helped us to reveal that posttranslational modifications (PTMs) on its N- and C-terminus have long-range impact on IDP structure such as HMGN-1 (up 60 amino acids away from the PTM). To extend our scope of PTMs and to target membrane-bound proteins as well, we generated a lipidated und isotopically labelled CD44 segment. CD44 is a multifunctional protein that resides on the cell surface and is involved in cell adhesion and migration but also in tumour formation. This lipidated CD44 will allow NMR studies of membrane-bound CD44 with a defined labelling pattern to study its structural changes upon membrane interaction and its interaction with binding partners. Our diverse protein targets clearly demonstrate that the structure zoom platform technology can be applied to very different protein targets (with respect to size, structure, localization etc.) for which zooming in on local structures is of high interest. We already obtained exciting new biochemical insights and will continue collaborating to gain more and to overcome delays caused by the Corona pandemic.