Structural Studies and Protein Engineering of Human O6-Alkylguanine-DNA Alkyltransferase

The specific labeling of proteins with synthetic probes is a powerful approach to study protein function and protein tags have been widely used for this purpose. A well-established example for a self-labeling protein tag is SNAP-tag. It specifically reacts with a wide variety of O6-benzylguanine derivatives (BG-derivatives) and was derived from the human O6-alkylguanine-DNA alkyltransferase (hAGT) by protein engineering. Relative to hAGT, SNAP-tag possesses a 52-fold higher reactivity towards BG-derivatives, does not bind to DNA and expresses well in cells as on cell surfaces. It is known that alkylation of hAGT results in protein unfolding and degradation. However, an increased degradation of SNAP-tag fusion proteins after labeling has not been observed. The first part of this work focused on the structural basis underlying the differences in protein stability between SNAP-tag and hAGT. A detailed biochemical and structural analysis was performed to determine (i) the interaction of SNAP-tag with its substrate, (ii) the factors responsible for its increased reactivity and (iii) how the labeling affected the stability of the protein. Besides an increased reactivity with BG-derivatives the superior stability of SNAP-tag compared to the parent protein hAGT could be confirmed. Whereas wild-type hAGT was rapidly degraded in cells after alkyl transfer, benzylated SNAP-tag showed a higher stability against proteolytic degradation. Moreover, the combination of our crystallographic and computational data provided further insight into the structural basis for the improved properties. The data indicated that the intrinsic stability of a key alpha helix was an important factor in triggering the unfolding and degradation of wild-type hAGT and provided new insights into the structure-function relationship of this DNA repair protein. The second part was aiming for the generation of a new SNAP-tag-based inhibitor complex. It was envisaged that this complex would interact with the target protein via amino acid loops and decrease its function only upon labeling with BG-inhibitor molecules. Therefore, SNAP-tag was modified by the insertion of stretches of randomized amino acids and the generated protein libraries were screened for binding affinity. The utilization of two yeast-based systems, the yeast three-hybrid and two hybrid technologies, allowed for the differentiation of small-molecule dependent and independent binding interactions. It could be demonstrated that a specific protein-loop interaction can be generated by this approach. It could further be shown that inhibition of the catalytic activity of the target protein E.coli dihydrofolate reductase by a SNAP-loop mutant was possible. In summary this work revealed new insights into the stability of hAGT and SNAP-tag and the structure-function relationship of AGTs in general. Further, SNAP-tag engineering generated a new protein-binder whose affinity towards the target protein was leading to protein inhibition.

A Biochemical and Biophysical Investigation of 5-HT3 Receptor Stability

Menno Berend Tol

The 5-hydroxytryptamine 3 receptor (5-HT3R) is a member of the pentameric ligand-gated ion channel (pLGIC) family, that plays an important role in fast signal transduction and cell-cell communication in synapses: They convert a chemical signal from a neurotransmitter into the electrical signal that represents the action potential. Activation of 5-HT3R with serotonin triggers a range of molecular reorganizations that lead to channel opening and subsequent membrane depolarization. Because of their role, pLGICs are involved in a wide range of diseases and disorders and are therefore important drug targets. A typical pLGIC is composed of a β -sheet rich extracellular domain (ECD) where ligand binding takes place, a transmembrane domain that is composed of four α-helices (TM1-TM4). In vertebrates pLGICs often contain a third intracellular domain (ICD), located between TM3 and TM4. The 5-HT3R is active as a homopentameric receptor of five identical A subunits, or as a heteropentamer containing one or more of several subunits (B to E) together with an A subunit. The 5-HT3 receptor is naturally located in the cell membrane in low amounts. For in vitro experiments such as biophysical investigation, the receptor has to be overproduced and purified in a detergent-solubilized state. Therefore, a robust and scalable expression system for homo- and heteropentameric 5-HT3 receptors was developed, building upon a tetracycline inducible cell line and Strep-tag affinity purification technology. Production of solubilized and purified 5-HT3R is well suited for use in the structural and functional characterization of the receptor. The stability of the 5-HT3 receptor is an important parameter during structural and functional experimentation. Using thermal unfolding the stability of 5-HT3R was investigated in plasma membranes as well as during detergent-extraction, purification and reconstitution into artificial lipid bilayers. A large loss in thermostability was found that correlates with the loss of the lipid bilayer during membrane solubilization and purification. Thermal unfolding of 5-HT3R occurred in consecutive steps at distinct protein locations: First a loss of ligand binding is detected, followed by formation of different transient low oligomeric states of receptor pentamers, followed by partial unfolding of helical parts of the protein, which finally leads to the formation large receptor aggregates. It was also found that structural destabilization of the receptor in detergents could be partially reversed by reconstituting the receptor into lipid bilayers. Thermal unfolding leads to the irreversible formation of aggregates which is known to prevent effective refolding. Therefore, an further investigation of 5-HT3R was made by unfolding the receptor in urea and sodium dodecyl sulfate (SDS). The results indicate iii iv ABSTRACT that, unlike thermal unfolding, unfolding in urea and SDS does not result in the formation of aggregates. Unfolding by urea shows a 50% loss of helical content while keeping the characteristic pentamer intact. Contrastingly, unfolding by SDS shows dissociation of the pentamer, but no noticeable change in secondary structure. Still, refolding from urea and SDS proved to be impossible. Two weak fluorophores, crystal violet (CrV) and ethidium bromide (EtBr), bind to the homologous nicotinic acetylcholine receptor (nAChR). Because the fluorescence of these compounds is also sensitive towards their environment they can be used as a molecular beacon for the functional state of a receptor. When applied to 5-HT3R, it was found that CrV and EtBr bind with micromolar affinity to the desensitized state of the receptor – similar to binding reported for the nAChR. It appeared that binding of CrV or EtBr to the 5-HT3R induces a certain functional state (resting, open or desensitized). Because no detectable change in fluorescence occurs, this prevents their use as molecular beacons. Still, non-competitive binding to the 5-HT3R was little reported on before and could be an important target in pharmacological research. The ICD of the 5-HT3R is predicted to contain a stretch of ∼60 disordered or flexible residues which might be involved in subunit assembly and membrane trafficking. These unstructured regions of a 5-HT3R were probed using limited proteolysis. Thereby two distinct fragments were formed that remain tightly associated with each other, while a small stretch of 35 residues between TM3 and TM4 was removed. Furthermore, it was shown that the thermal stability of the helical core is unchanged compared to untreated 5-HT3R, but that the extracellular ligand-binding domain is very sensitive to cleavage in the ICD. By probing the 5-HT3R with limited proteolysis a rigid core was identified, indicating the important role of the transmembrane domain for holding together the pLGIC scaffold. The results presented here contribute a significant body of knowledge to membrane proteins in general and to the 5-HT3 receptor in particular, upon which further research can be based leading to advances in the fields of biochemistry, biophysics and pharmacology.

EPFL2012

Structural characterisation and membrane insertion of M13 procoat, M13 coat and Pf3 coat proteins

Membrane proteins fulfill many central functions in the biological membrane. The insertion process of these proteins and their structure, which are intimately linked to their function, are not yet well understood. As a model we studied three proteins reconstituted into lipid bilayers (which serve as a model for the biological membrane): the major coat proteins of phages Pf3 and M13 and the precursor of the latter, M13 procoat protein, which spans the inner membrane two times and serves as a model for a multi-spanning membrane protein. Whereas Pf3 coat protein can insert into the membrane in its mature form, M13 coat protein needs the signal sequence present in the precursor to insert. A new, simple and efficient purification method has been developed for M13 procoat, M13 and Pf3 coat proteins. Homogenous preparations were obtained in isopropanol/0.l% TFA, where M13 coat protein is found to be dissolved in a monomeric form, and the two other proteins as dimers. The conformations of these particular proteins in different environments have been determined by circular dichroism and infrared spectroscopy. In organic solvents, the proteins adopt a conformation with an average helix content of 90%. In lipid bilayers composed of phospatidylcholine and phosphatidylglycerol lipids, the average helix content is 50% for M13 procoat protein, 60% for M13 coat protein and 75% for Pf3 coat protein. In order to characterize the orientational distribution the orientational order parameter Sα of the protein helices in planar lipid bilayers have been determined by polarized infrared measurements in the amide I spectral range. The helices of the three proteins are oriented preferentially parallel to the membrane normal, with Sα = 0.63 for M13 procoat protein, Sα = 0.58 for Pf3 coat protein and a distinctly higher value of Sα = 0.81 for M13 coat protein. The topology of M13 and Pf3 coat proteins was investigated using limited proteolysis in lipid vesicles with proteinase K combined with mass spectroscopy to analyze the lytic fragments. The C-terminal part of both proteins was found to be inaccessible to proteinase K, only 14 (Pf3) or 15 (M13) residues on the N-terminal were protease accessible from the outside of the vesicles. Furthermore, the standard free energy change, ΔGo, of a membrane-inserting protein with a leader sequence has been determined experimentally for the first time, using M13 procoat protein as an example. The partition coefficient for the distribution of this protein between the aqueous phase and the membrane phase of preformed lipid vesicles yielded a value of Γ = 6.5×105 M-1, corresponding to a ΔGo of -10.4 kcal/mol, based on measurements of the fluorescence energy transfer between the intrinsic tryptophan of the protein and a suitably labeled lipid membrane of POPC. For comparison, the partition coefficient of the M13 coat protein between the aqueous and the POPC lipid bilayer phase was determined to be distinctly lower: Γ = 1×105 M-1 (ΔGo = -9.3 kcal/mol). Proteinase K digestion experiments have been performed, showing that 20% of the procoat protein bound to lipid vesicles spontaneously integrate in a transbilayer form, whereas 80% remain inserted in the interfacial membrane region. By taking together these results, an upper limit for the free energy change of the transmembrane insertion of procoat protein was estimated to be -14.8 kcal/mol. In order to distinguish further the contribution arising from insertion of the procoat protein into the membrane interfacial region from that due to transmembrane insertion, the partition coefficient of a mutant procoat protein OM30R (which contains a positively charged amino acid in its mature hydrophobic segment (exchange of a Val to an Arg residue at position 30)) was determined, yielding Γ = 0.3×105 M-1 (ΔGo = -8.6 kcal/mol). Previously reported in vivo experiments have shown that the OM30R mutant protein is not translocated across Escherichia coli inner membrane, but only binds to the inner surface. The results presented here indicate that although the insertion of the procoat protein into the interfacial region of the lipid bilayer contributes the major part to ΔGo, it is the final energy gain of the interaction of the hydrophobic portions of the folded pre-protein with the lipid chains which drives the transmembrane insertion of the M13 procoat protein. Neither the leader sequence nor the mature coat protein alone yields this free energy gain. For the different proteins investigated here, spontaneous membrane insertion occurs only for fluid lipid bilayers, but not for membranes in the crystalline lipid phase. Furthermore, by using lipid bilayers with negative membrane surface charges, it was shown that both procoat and coat proteins are electrostatically attracted to the surface of the lipid membrane, though only to a small extent, with apparent partition coefficients of the same order of magnitude as for the phosphatidylcholine lipid membrane. This work has yielded new information about the structure and the insertion of the phage coat proteins. The new methods developed in this work might be of general applicability to study the structural dynamics of membrane protein assembly.

EPFL1996

In Vivo Studies on the Control of Gene Expression and Protein Degradation

Karolina Bojkowska

Cellular homeostasis is maintained by tightly regulated gene networks, controlled notably at the levels of transcription, mRNA processing, and protein post-translational modification and turnover. This thesis focuses on two of these regulatory steps, namely gene transcription and protein stability. I first explored the impact of the KRAB-ZFP/KAP1 gene regulation system on liver function. KRAB-ZFPs constitute the largest family of transcription factors encoded by mouse and humans, yet their physiological roles and gene targets remain mostly elusive. Conversely, their molecular mechanism of action has been rather well characterized, as they can recognize DNA in a sequence-specific manner and recruit the universal co-factor KAP1 (also known as TRIM28), which in turn serves as a scaffold for various heterochromatin-inducing effectors. Several studies have shed some light on the roles played by this system in embryonic tissues, but its functions in the adult remain almost completely unknown. As chromatin regulation has been shown to be a major factor in the control of metabolism, we decided to investigate the role of KRAB-ZFP/KAP1 in the liver, an organ central to this process. For this, we generated conditional liver-specific Kap1 KO mice, the study of which revealed a strikingly sex-specific phenotype, with early-onset steatosis and age-related tumorigenesis restricted to males, contrasting with the mild metabolic disturbances recorded in Kap1-deleted females. Underlying this phenotype, our combined transcriptome and chromatin analyses revealed that KAP1 controls sexually dimorphic genes notably involved in hormone, drug and xenobiotic metabolism. Finally, by using a recently developed technique for direct RNA quantification, we established the range of KRAB-ZFPs expressed in the liver and likely responsible for at least part of the observed effects. This study thus demonstrates the central role of the KRAB/KAP1 system in control of sexual dimorphism, responses to xenobiotic stress and metabolic control in the liver, and further links disturbances in these processes with hepatic carcinogenesis. In parallel, in an effort to pursue a multidisciplinary training combining skills in biology and chemistry, I worked towards the development of a method to study the half-life of proteins in vivo. Protein turnover critically influences many biological functions, yet methods have been lacking to assess this parameter in vivo. Capitalizing on an in vitro technology previously developed in Kai Johnsson's laboratory, I demonstrated how chemical labeling can be used to measure the half-life of resident intracellular and extracellular proteins in living mice. Our approach is based on labeling of SNAP-tag-fusion proteins with fluorescent probes. First, we verified that SNAP-tag substrates have wide bio-availability in mice and can be used for the specific in vivo labeling of SNAP-tag fusion proteins. We then applied near-infrared probes to perform non-invasive imaging of in vivo labeled tumors. Finally, we used SNAP-mediated chemical pulse-chase labeling to measuring in vivo the half-life of different extra- and intracellular proteins, including KAP1. Importantly, this tagging technology can be applied to any protein amenable to expression as a fusion partner, whether cell-associated or secreted. Furthermore, because the SNAP-tag chemical labeling allows for a large array of probes also suited for experiments aimed at manipulating and monitoring protein function in vivo, such as cross-linking, pull-down or FRET, this methodology opens broad perspectives for studying protein function in living animals.

EPFL2011