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Proteins are central agents for all cellular processes of life. They confer shape or enable motility, act as defence or a means of aggression, and catalyse a plethora of chemical reactions. A protein’s function is typically determined by its three-dimensional structure and, indeed, many human diseases arise when proteins misfold or denature prematurely. Yet life has continuously evolved since its inception, implying that there are underlying principles governing which changes are viable and which are not. By determining the atomic structure of proteins, we can follow these evolutionary trajectories and unveil how they influence biological function. With cutting-edge scientific methods, such insights can, in turn, be used to guide the design of new drugs, or, due to recent advances in engineering, the synthetic design of entirely novel proteins. 

In this thesis, I aim to contribute to the fundamental understanding of structural biology by exploring aspects of both natural and directed protein evolution. I used state-of-the-art cryo-Electron Microscopy (cryo-EM) to characterize protein structures, enabling detailed analyses of how evolution navigates structural constraints to repurpose protein folds and optimize molecular interactions. 

In the first project of this thesis, I investigate the emergence of novel ribosomal proteins in Microsporidia. By analysing high-resolution structural models of their ribosomes, I observed a progressive reduction of ribosomal RNA, loss of expansion segments, and truncation of ribosomal proteins. To maintain ribosomal integrity, microsporidia acquired new proteins that compensate for the loss of stabilizing interactions. I discovered that these proteins were in essence ‘evolved fold-switchers’: homologs that adopted highly dissimilar structures, with inverted termini, despite occupying the same ribosomal niche. Thus, these findings represent a rare example where a proteins’ function and cellular environment are more conserved than its fold. 

The second project shifts the focus towards directed evolution. Here, an affinity protein, CaRAEGFR, was engineered to recognize the cancer marker epidermal growth factor receptor (EGFR) via a mechanism calibrated to high calcium concentrations typically found in the bloodstream. As a result, CaRAEGFR bind its target with high affinity whilst in circulation but loses this affinity after being trafficked into cells via endocytosis. This conditionally regulated mechanism greatly improved CaRAEGFRs potential to act as a targeting unit for protein-drug conjugates, and we demonstrated potent cytotoxicity in EGFR-expressing cancer cells when CaRAEGFR was coupled to a drug. Although the complex of CaRAEGFR and EGFR corresponds to a size of only ~80 kDa, I could confirm their interaction using single-particle analysis (SPA) cryo-EM. 

In the third project, I expand on these findings by providing a more detailed structural characterization of the CaRAEGFR:EGFR complex using cryo-EM. I observed that CaRAEGFR engaged with EGFR via a hydrophobic cleft in domain III and compared this mode of recognition to other known EGFR-binding proteins. Complementary NMR experiments were used to probe calcium-dependent structural changes, allowing us to propose an allosteric mechanism in which environmental calcium concentrations modulate this interaction. 

The fourth project returns to natural protein evolution. Here, I investigated the biochemical properties of BmdE, a nuclease involved in interspecies biofilm dispersion. Using cryo-EM, I solved the structure of its nuclease core, revealing a novel, extended phosphodiesterase domain comprising an a/b sandwich motif. Notably, the cryo-EM data contained additional densities suggesting an intermediate reaction state ‘trapped’ after DNA cleavage. The structural information was then used to establish an evolutionary context, revealing that BmdE-like nucleases represent a kingdom-spanning protein family rather than a newly evolved, species-specific mechanism. 

In summary, this thesis explores diverse structure-function relationships across evolutionary scales, ranging from the de novo birth and subsequent fold switching of ribosomal proteins to a rationally engineered, conditionally regulated affinity protein for cancer treatment. Collectively, this work contributes new insights into the adaptability of protein architectures that may guide future efforts in structure-based drug design, protein engineering and biofilm control. 

Proteiner är centrala aktörer i alla cellers livsprocesser. De ger form eller möjliggör motilitet, fungerar som försvar eller som ett medel för angrepp och katalyserar en mängd reaktioner, som exempelvis energiomvandling. Ett proteins funktion bestäms vanligtvis av dess tredimensionella struktur, och många sjukdomar hos människan uppstår faktiskt när proteiner veckas fel, denatureras eller muterar. Ändå har livet kontinuerligt utvecklats sedan dess uppkomst, vilket antyder att det finns underliggande principer som styr vilka förändringar som är livskraftiga och vilka som inte är det. Genom att bestämma proteiners atomstruktur kan vi följa dessa evolutionära utvecklingsbanor och förklara hur de påverkar dess molekylära funktion. Med hjälp av banbrytande vetenskapliga metoder kan sådana insikter i sin tur användas för att vägleda utformningen av nya läkemedel, eller, i allt högre grad, den syntetiska designen av helt nya proteiner genom ingenjörskonst. 

Denna avhandling syftar till att öka förståelsen för de grundläggande principerna inom strukturbiologin genom att utforska aspekter av både naturlig och styrd proteinevolution. Avancerad kryo-EM har använts för att strukturellt karakterisera två exempel på ovanligt små proteiner, vilket möjliggör detaljerade analyser av hur evolutionen hanterar strukturella begränsningar för att omforma proteinstrukturer och optimera molekylära interaktioner. 

Det första projektet i denna avhandling undersöker uppkomsten av nya ribosomala proteiner hos mikrosporidier. Genom att analysera högupplösta strukturmodeller av deras ribosomer kunde vi observera en gradvis reducering av rRNA, förlust av expansionssegment samt en förkortning av ribosomala proteiner. För att upprätthålla ribosomernas integritet förvärvade mikrosporidierna nya proteiner som kompensation för förlusten av stabiliserande interaktioner. Vi upptäckte att dessa proteiner i huvudsak var ”evolutionära veckningsväxlare” – homologer som antog väldigt annorlunda strukturer med inverterade terminaler, trots att de upptog samma ribosomala nisch. Dessa fynd representerar ett sällsynt exempel där ett proteins funktion och cellulära miljö är mer bevarade än dess veckning. 

Det andra projektet fokuserar på riktad evolution. Det beskriver utvecklingen av ett affinitetsprotein, CaRAEGFR, som utvecklats för att binda cancermarkören EGFR via en kalciumskoncentrationsberoende mekanism. Genom att göra proteinets aktivitet kalciumberoende kunde den skräddarsys för att binda sitt mål medan den cirkulerar, men också snabbt dissocieras efter internalisering. Detta förbättrade dess potential som ett mål för protein-läkemedelskonjugat avsevärt; vi visade också hög cytotoxicitet i EGFR-uttryckande cancerceller när bindaren kopplades till ett läkemedel. Även om komplexet av bindare och receptor endast motsvarar ~80 kDa, kunde vi initialt bekräfta deras interaktion med hjälp av kryo-elektronmikroskopi. 

Det tredje projektet bygger vidare på dessa resultat genom att tillhandahålla en mer detaljerad strukturell karakterisering av bindningsmolekylen i komplex med sin receptor. Med hjälp av kryo-EM observerade vi att CaRAEGFR binder till EGFR via en hydrofobisk hotspot i domän III och jämförde detta bindningssätt med andra kända EGFR-bindande proteiner. Kompletterande NMR-experiment användes för att undersöka kalciumberoende strukturella förändringar, vilket gjorde det möjligt för oss att föreslå en allosterisk mekanism genom vilken kalciumkoncentrationer i omgivningen modulerar denna interaktion. 

Det fjärde projektet återvänder till den naturliga evolutionen. Här undersökte jag de biokemiska egenskaperna hos ett nukleas som är inblandat i nedbrytning av biofilm. Med hjälp av kryo-EM löste vi strukturen hos nukleasets-kärna, vilket avslöjade en ny, utsträckt fosfodiesterasdomän bestående av ett α/β-sandwichmotiv som inte tidigare har associerats med extracellulär DNA nukleaser. Noterbart är att kryo-EM data innehöll ytterligare densiteter som tyder på en reaktionsmellanprodukt som ”fastnat” under DNA-klyvningen. Strukturinformationen användes sedan för att fastställa ett evolutionärt sammanhang, vilket avslöjade en proteinfamilj som spänner över flera riken snarare än en unikt utvecklad mekanism. 

Sammanfattningsvis undersöker denna avhandling olika struktur-funktionssamband på olika evolutionära nivåer, från ribosomproteiners uppkomst från grunden och efterföljande omformning till rationell design som möjliggör kontrollerad reglering. Sammantaget bidrar detta arbete med nya insikter om proteinarkitekturens anpassningsförmåga, vilket kan ligga till grund för framtida insatser inom strukturbaserad läkemedelsdesign, proteinmodifiering och bekämpning av biofilmer.

The emerging strategy of protein–drug conjugates (PDCs) for targeted cancer therapy holds great potential to improve treatment efficacy by specifically targeting cancer biomarkers and delivering toxic payloads directly to tumor cells, minimizing off-target toxicity. The success of this approach depends on the internalization and retention of the payload in target cells. This study introduces a method using a small protein domain engineered for conditional target affinity, enabling lysosomal trafficking independent of the biological fate of the receptor. Specifically, we describe the development of an EGF receptor binder, CaRAEGFR, with calcium-regulated affinity (CaRA), meaning the target binding strength is tailored by the available calcium concentration. This allows for endosomal dissociation, as calcium levels are lower in endosomes than in the bloodstream. Affinity measurements and structural modeling reveal the molecular basis of the calcium modulated affinity. Live cell imaging demonstrates efficient internalization and lysosomal trafficking of the calcium-dependent domain, while the EGF receptor is recycled to the membrane. When used as a drug carrier, CaRAEGFR effectively delivers the toxin to the lysosomes, resulting in potent cytotoxicity with an IC50 of 0.8 nM in EGFR-expressing cancer cells

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.

Ribosomes from different species can markedly differ in their composition by including dozens of ribosomal proteins that are unique to specific lineages but absent in others. However, it remains unknown how ribosomes acquire new proteins throughout evolution. Here, to help answer this question, we describe the evolution of the ribosomal protein msL1/msL2 that was recently found in ribosomes from the parasitic microorganism clade, microsporidia. We show that this protein has a conserved location in the ribosome but entirely dissimilar structures in different organisms: in each of the analyzed species, msL1/msL2 exhibits an altered secondary structure, an inverted orientation of the N-Termini and C-Termini on the ribosomal binding surface, and a completely transformed 3D fold. We then show that this fold switching is likely caused by changes in the ribosomal msL1/msL2-binding site, specifically, by variations in rRNA. These observations allow us to infer an evolutionary scenario in which a small, positively charged, de novo-born unfolded protein was first captured by rRNA to become part of the ribosome and subsequently underwent complete fold switching to optimize its binding to its evolving ribosomal binding site. Overall, our work provides a striking example of how a protein can switch its fold in the context of a complex biological assembly, while retaining its specificity for its molecular partner. This finding will help us better understand the origin and evolution of new protein components of complex molecular assemblies-thereby enhancing our ability to engineer biological molecules, identify protein homologs, and peer into the history of life on Earth.

Phosphoryl transfer is a fundamental reaction in cellular signaling and metabolism that requires Mg2+ as an essential cofactor. While the primary function of Mg2+ is electrostatic activation of substrates, such as ATP, the full spectrum of catalytic mechanisms exerted by Mg2+ is not known. In this study, we integrate structural biology methods, molecular dynamic (MD) simulations, phylogeny, and enzymology assays to provide molecular insights into Mg2+-dependent structural reorganization in the active site of the metabolic enzyme adenylate kinase. Our results demonstrate that Mg2+ induces a conformational rearrangement of the substrates (ATP and ADP), resulting in a 30° adjustment of the angle essential for reversible phosphoryl transfer, thereby optimizing it for catalysis. MD simulations revealed transitions between conformational substates that link the fluctuation of the angle to large-scale enzyme dynamics. The findings contribute detailed insight into Mg2+ activation of enzymes and may be relevant for reversible and irreversible phosphoryl transfer reactions.