Structural Analysis of Protein Complexes

Abstract

A. Mitochondria travel over long distances to their sites of function to meet local energy requirements and maintain calcium homeostasis. Alterations in mitochondrial distribution in neurons are closely linked to neurodegenerative diseases such as Parkinson’s disease. Key regulators of mitochondrial trafficking are the calcium-sensing GTPase Miro and the motor adaptor protein TRAK, which couple mitochondria to the motor proteins. Despite increasing evidence for the role of Miro in many cellular processes including mitochondrial distribution, dynamics and turn-over, the underlying mechanistic details remain elusive. Understanding the conformational changes Miro might undergo upon calcium-binding, GTP hydrolysis or protein-protein interactions is the focus of this study. For this purpose, we established an expression and purification protocol for the two human Miro proteins, namely hMiro1 and hMiro2. hMiro1 was characterised in terms of stability, folding and for the first time regarding the GTPase activity in context of the full-length protein. Furthermore, we implemented the expression and purification of the hMiro1 binding site of hTRAK2, one of the two human TRAK proteins. We were able to form a complex between hMiro1 and hTRAK2 demonstrating that recombinant hMiro1 is functional. The hMiro1 purified in this study has been used by Funmi Fagbadebo (group of Prof. Rothbauer, Pharmaceutical Biotechnology, University of Tübingen) to immunise alpacas and to select, enrich and characterise hMiro1-specific nanobodies obtained from corresponding blood samples. The established protocol is applicable to Parkinon’s disease-derived mutations of hMiro1 thus enabling important future research. Taken together, this project provides a platform for the investigation of the structure-function relationship of hMiro1 and its interacting partners.

B. Bromodomains (BD) are unique structural modules that specifically recognise N-ε-lysine acetylation motifs. BD-containing proteins of the Bromodomain and Extra-Terminal domain (BET) family share a common domain architecture comprising two structurally conserved BDs. They serve as transcriptional regulators and epigenetic markers by association with acetylated histones throughout the cell cycle. Malfunction of BET proteins has been linked to a broad spectrum of diseases. BRD4, a member of this protein family, has been shown to be responsible for the regulation of growth-promoting genes in chronic lymphocytic leukaemia and has been identified as component of chromosomal translocation in nuclear protein in testis midline carcinoma. Despite the importance of studying these proteins, one major obstacle remains the deacetylation of BD targets in in vivo experiments. In 2010 Filippakopoulos et al. published a BET family specific, cell-permeable small molecule BD inhibitor, called JQ1, based on which Sören Kirchgäßner (group of Prof. Schwarzer, Interfaculty Institute of Biochemistry, University of Tübingen) designed the acetyl lysine mimicking artificial amino acid ApmTri used in this study. Affinity measurements and crystallisation experiments showed that BET BDs bind peptides comprising ApmTri with comparable affinity, high specificity and in a similar structural fashion compared to peptide substrates comprising acetyl lysine. We thereby introduce a new and versatile tool for the utmost important in vivo research on BD-containing proteins. We have built a solid foundation for the future design of peptide-based co-inhibitors, capable of inhibiting both BDs of BET family proteins at once, thus increasing the affinity and selectivity of the inhibitors.

C. Human adenoviruses (HAdV) are the cause for many respiratory, ocular and gastrointestinal diseases. They shield their double-stranded DNA core with an icosahedral capsid formed by three major proteins, the hexon, the fibre and the penton base. For virus entry, the major capsid proteins need to interact with host cell receptors to trigger virus internalisation. While the fibre knob is well-established as the interaction partner for primary receptors and the penton base is known to engage with integrins, recent data suggest a role for the hexon protein in receptor interaction. Based on infection data the interaction between the complement regulatory protein CD46 and the HAdV-D species was established, but additional research will be required to assess if the hexon is responsible for the interaction beyond the two genotypes that were investigated, HAdV D56 and HAdV D26. In order to elucidate which epitopes in the hyper variable region of the trimeric hexon protein engage in CD46 binding, we aim to obtain structural data on HAdV D type hexons in complex with CD46. For this purpose, we established complex formation with five different D-species hexons and optimised the hexon to receptor ratio to improve sample quality. Initial negative staining images of the HAdV receptor complex highlight the quality of the hexon sample and reveal the prevalent hexon orientation for each genotype. With these experiments we have laid the foundations for subsequent cryoEM experiments. The soon to be obtained structures will broaden our understanding of HAdV receptor interaction and help elucidating the potential of new HAdV genotypes as vaccine vectors.