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1.
Bioconjug Chem ; 34(6): 1114-1121, 2023 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-37246906

RESUMO

Enzymes are of central importance to many biotechnological and biomedical applications. However, for many potential applications, the required conditions impede enzyme folding and therefore function. The enzyme Sortase A is a transpeptidase that is widely used to perform bioconjugation reactions with peptides and proteins. Thermal and chemical stress impairs Sortase A activity and prevents its application under harsh conditions, thereby limiting the scope for bioconjugation reactions. Here, we report the stabilization of a previously reported, activity-enhanced Sortase A, which suffered from particularly low thermal stability, using the in situ cyclization of proteins (INCYPRO) approach. After introduction of three spatially aligned solvent-exposed cysteines, a triselectrophilic cross-linker was attached. The resulting bicyclic INCYPRO Sortase A demonstrated activity both at elevated temperature and in the presence of chemical denaturants, conditions under which both wild-type Sortase A and the activity-enhanced version are inactive.


Assuntos
Aminoaciltransferases , Proteínas de Bactérias , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Aminoaciltransferases/metabolismo , Peptídeos , Cisteína Endopeptidases/metabolismo
2.
J Biol Chem ; 295(52): 18266-18275, 2020 12 25.
Artigo em Inglês | MEDLINE | ID: mdl-33109610

RESUMO

Aminopeptidase N (APN, CD13) is a transmembrane ectopeptidase involved in many crucial cellular functions. Besides its role as a peptidase, APN also mediates signal transduction and is involved in the activation of matrix metalloproteinases (MMPs). MMPs function in tissue remodeling within the extracellular space and are therefore involved in many human diseases, such as fibrosis, rheumatoid arthritis, tumor angiogenesis, and metastasis, as well as viral infections. However, the exact mechanism that leads to APN-driven MMP activation is unclear. It was previously shown that extracellular 14-3-3 adapter proteins bind to APN and thereby induce the transcription of MMPs. As a first step, we sought to identify potential 14-3-3-binding sites in the APN sequence. We constructed a set of phosphorylated peptides derived from APN to probe for interactions. We identified and characterized a canonical 14-3-3-binding site (site 1) within the flexible, structurally unresolved N-terminal APN region using direct binding fluorescence polarization assays and thermodynamic analysis. In addition, we identified a secondary, noncanonical binding site (site 2), which enhances the binding affinity in combination with site 1 by many orders of magnitude. Finally, we solved crystal structures of 14-3-3σ bound to mono- and bis-phosphorylated APN-derived peptides, which revealed atomic details of the binding mode of mono- and bivalent 14-3-3 interactions. Therefore, our findings shed some light on the first steps of APN-mediated MMP activation and open the field for further investigation of this important signaling pathway.


Assuntos
Proteínas 14-3-3/metabolismo , Antígenos CD13/metabolismo , Metaloproteinases da Matriz/metabolismo , Proteínas 14-3-3/química , Motivos de Aminoácidos , Sítios de Ligação , Antígenos CD13/química , Cristalografia por Raios X , Ativação Enzimática , Humanos , Metaloproteinases da Matriz/química , Conformação Proteica , Transdução de Sinais
3.
Angew Chem Int Ed Engl ; 58(48): 17351-17358, 2019 11 25.
Artigo em Inglês | MEDLINE | ID: mdl-31539186

RESUMO

Protein complex formation depends on the interplay between preorganization and flexibility of the binding epitopes involved. The design of epitope mimetics typically focuses on stabilizing a particular bioactive conformation, often without considering conformational dynamics, which limits the potential of peptidomimetics against challenging targets such as transcription factors. We developed a peptide-derived inhibitor of the NF-Y transcription factor by first constraining the conformation of an epitope through hydrocarbon stapling and then fine-tuning its flexibility. In the initial set of constrained peptides, a single non-interacting α-methyl group was observed to have a detrimental effect on complex stability. Biophysical characterization revealed how this methyl group affects the conformation of the peptide in its bound state. Adaption of the methylation pattern resulted in a peptide that inhibits transcription factor assembly and subsequent recruitment to the target DNA.


Assuntos
Fator de Ligação a CCAAT/química , Peptídeos/química , Multimerização Proteica/efeitos dos fármacos , Sequência de Bases , Sítios de Ligação , Reagentes de Ligações Cruzadas/química , Cristalização , DNA/química , Epitopos/química , Humanos , Compostos Macrocíclicos/química , Metilação , Simulação de Dinâmica Molecular , Peptidomiméticos , Ligação Proteica , Conformação Proteica , Termodinâmica
4.
Chem ; 10(2): 615-627, 2024 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-38344167

RESUMO

Proteins are essential biomolecules and central to biotechnological applications. In many cases, assembly into higher-order structures is a prerequisite for protein function. Under conditions relevant for applications, protein integrity is often challenged, resulting in disassembly, aggregation, and loss of function. The stabilization of quaternary structure has proven challenging, particularly for trimeric and higher-order complexes, given the complexity of involved inter- and intramolecular interaction networks. Here, we describe the chemical bicyclization of homotrimeric protein complexes, thereby increasing protein resistance toward thermal and chemical stress. This approach involves the structure-based selection of cross-linking sites, their variation to cysteine, and a subsequent reaction with a triselectrophilic agent to form a protein assembly with bicyclic topology. Besides overall increased stability, we observe resistance toward aggregation and greatly prolonged shelf life. This bicyclization strategy gives rise to unprecedented protein chain topologies and can enable new biotechnological and biomedical applications.

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