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Proteins are the workhorses of the cell and support all cellular functions governing biological life. The diversity of protein function is intimately linked to the unique threedimensional structure and intrinsic dynamics of each individual protein. One class of proteins, kinases, are enzymes that catalyze the transfer of a phosphoryl group from ATP to substrate molecules, thereby playing pivotal roles in cellular processes. There are different types of kinases acting on substrates ranging from a simple nucleotide to an entire protein. Aurora B  is a protein kinase with essential roles in cell division and is regulated by multiple mechanisms that ensure faithful mitotic progression. Adenylate kinase (AK) is a small nucleotide kinase that maintains the energy homeostasis in the cell by interconverting ATP and AMP into two ADP molecules.  In this thesis, the link between function, dynamics and structure has been explored in the two kinases Aurora B and AK by combining a selection of structural and functional studies. 

Studies on Aurora kinase B were focused on overcoming production limitations and exploring the effects of phosphorylation on protein dynamics and function. In Paper I, we developed a new production approach of human Aurora B in complex with its regulatory partner inner centromere protein (INCENP) in order to address previous production limitations. The developed approach was based on a fusion protein design with robust expression levels in E. coli, resulting in pure and fully functional protein with yields on the mg-scale. This approach enabled further structural and functional studies and was used to study the effects of phosphorylation on dynamics and substrate binding in Paper II. In this latter paper, we conducted a comparative study between the phosphorylated and dephosphorylated Aurora B:INCENP complex by probing protein dynamics with ¹⁹F NMR spectroscopy experiments. We found that the complex exists in a conformational equilibrium between inactive and active structural states, which is greatly influenced by phosphorylation and substrate binding.

The studies of AK aimed at understanding how small structural elements, such as termini of α-helices and protein bound water molecules, possibly facilitate catalysis. In Paper III, we investigated whether the termini of α-helices could support the large-scale conformational changes underlying catalysis in AK. We employed a comparative approach focused on a bacterial, archaeal and human AK, and found through a combination of X-ray crystallography, NMR spectroscopy and enzymatic activity assay experiments, that the inherent flexibility in the termini of α-helices influences the conformational changes correlated to catalysis. In Paper IV, the role of water molecules in AK catalysis was investigated by swapping the solvent from water to deuterium oxide and measure the effects on catalysis, substrate binding and protein stability with NMR spectroscopy, ITC and CD spectroscopy experiments. We showed that water molecules have an impact on AK catalysis, structural stability and coordination of indirect substrate contacts, all combined suggesting an indirect but important role of water molecules in the phosphoryl transfer reaction. Overall, this thesis contributes to an advanced understanding of how protein structure and dynamics modulate the catalytic function of kinases. 

Yersinia pathogenicity is dependent on polarized translocation of effectorproteins via the type III secretion system (T3SS). The tip complex situatedon the needle structure of the T3SS is required for contact with the eukaryotichost membrane and is to an extent composed of pentameric LcrV. LcrVis a multifunctional protein that also acts as a regulator of the T3SS by virtueof forming a high-affinity complex in the cytoplasm with its chaperone, LcrG.By employing a structure-based approach centered on mass spectrometry,FRET and NMR spectroscopy, we demonstrated that the LcrV-LcrG complexis best described as a multivalent complex, and that the N-terminaldomain of LcrV contributes by negatively affecting the LcrG binding affinity.The N-terminal domain of LcrV is dynamic and undergoes a conformationalchange to accommodate LcrG binding. 19F NMR spectroscopy analysissuggests that the conformational change is an intrinsic property of the protein,which agrees with a conformational selection model. An analysis ofeffector secretion into a culture supernatant demonstrated that the low synthesisand low secretion phenotypes of a LcrV mutant where the N-terminaldomain has been removed are linked to the structure, interactions and stabilityof the LcrV N-terminal domain. In summary, our results add insightsinto the dynamics of LcrV and its complex with LcrG.

Conformational dynamics is a fundamental aspect of enzymatic catalysis that, for example, can be linked to ligand binding and release, assembly of the active site, and the catalytic mechanism. The essential and metabolic enzyme adenylate kinase (AK) undergoes large-scale conformational changes in response to binding of its substrates ATP and AMP. As such, it has been intensely studied in search of linkages between dynamics and catalysis. For a complex conformational change to occur in a protein, whether it is of an induced fit or conformational selection nature, changes at several hinges are often required. Here, based on a comparative structure–function analysis of AK enzymes from E. coli and the archaea Odinarchaeota and from human AK1, we found that conformational changes in the enzymes are to a varying degree linked to bending, fraying, or unfolding/folding events of the termini of α-helices observed in various structural hot spots of the enzymes. The findings contribute with a mechanistic angle to how enzymatic dynamics and catalysis relate to the plasticity of the termini of α-helices.

Protein kinases are key players in many eukaryotic signal transduction cascades and are as a result often linked to human disease. In humans, the mitotic protein kinase family of Aurora kinases consist of three members: Aurora A, B and C. All three members are involved in cell division with proposed implications in various human cancers. The human Aurora kinase B has in particular proven challenging to study with structural biology approaches, and this is mainly due to difficulties in producing the large quantities of active enzyme required for such studies. Here, we present a novel and E. coli-based production system that allows for production of milligram quantities of well-folded and active human Aurora B in complex with its binding partner INCENP. The complex is produced as a continuous polypeptide chain and the resulting fusion protein is cleaved with TEV protease to generate a stable and native heterodimer of the Aurora B:INCENP complex. The activity, stability and degree of phosphorylation of the protein complex was quantified by using a coupled ATPase assay, 31P NMR spectroscopy and mass spectrometry. The developed production system enables isotope labeling and we here report the first 1H–15N-HSQC of the human Aurora B:INCENP complex. Our developed production strategy paves the way for future structural and functional studies of Aurora B and can as such assist the development of novel anticancer drugs targeting this important mitotic protein kinase.