Estudo estrutural do domínio SH3-like ligado em tandem da proteína KIN humana e sua interação com ácido nucleico utilizando Espectroscopia de Ressonância Magnética Nuclear e caracterização físico-química por técnicas espectroscópicas
Data
2023-01-27
Autores
Lourenço, Isabella Otenio de
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Universidade Estadual Paulista (Unesp)
Resumo
A proteína KIN atua como reguladora de danos no DNA e está envolvida na replicação desse ácido nucleico. Seu gene, localizado no cromossomo 10 em células humanas, é filogeneticamente conservado e ubiquamente expresso em eucariotos. Em humanos, essa proteína possui 45 kDa e é estruturalmente composta por quatro domínios, sendo: domínio dedo de zinco do tipo C2H2, domínio central homólogo à proteína RecA de E. coli, domínio de sinal de localização nuclear e domínio SH3-like em tandem (dSH3thKIN) formado por dois subdomínios SH3. Estudos mostraram que o dSH3thKIN apresenta em sua estrutura um motivo de ligação a RNA conhecido como motivo KOW e, portanto, esse domínio é considerado como o principal mediador de interação da proteína KIN com RNA. Essa interação dSH3thKIN/RNA é fundamental para o desempenho das atividades biológicas da KIN. Entretanto, há uma escassez de informações estruturais e de interação do dSH3thKIN na literatura. Dessa forma, o presente trabalho teve como objetivo realizar o assinalamento das ressonâncias 1H, 15N e 13C do domínio, determinar sua estrutura tridimensional em solução, caracterizar a estabilidade térmica, química e de pH desse domínio e investigar sua interação com RNA de levedura e homopolímeros de RNA e DNA. Um total de 98,4% das ressonâncias do domínio foram assinaladas e a resolução de sua estrutura tridimensional apresentou dois subdomínios com arranjos de barris-β formados por cinco fitas-β cada um. A análise de movimentos térmicos da cadeia principal do dSH3thKIN mostrou que esse domínio apresenta uma flexibilidade interna reduzida apesar de ser formado por dois subdomínios. A caracterização da estabilidade térmica e de pH do domínio revelou uma temperatura de desenovelamento de aproximadamente 59 oC, com uma tendência de agregação que leva a formação de estruturas como amiloides, e uma estabilidade das estruturas secundárias entre pH 5,5 e 9,5. O processo de desenovelamento químico do dSH3thKIN ocorreu de modo reversível, sugerindo que esse domínio apresenta um enovelamento independente no contexto da proteína KIN. As medidas de interação do domínio com ácidos nucleicos mostraram a seguinte ordem de afinidade: poli(rG) > poli(rC) > RNA de levedura > poli(rU) > poli(rA) > poli(A) DNA, indicando que o dSH3thKIN apresenta uma maior afinidade pelas bases de guaninas e citosinas e RNA de levedura. A análise dos parâmetros termodinâmicos da ligação dSH3thKIN/RNA sugere que a maior contribuição para a formação do complexo é a interação eletrostática. Detalhes estruturais sobre o sítio de ligação de ácido nucleico no dSH3thKIN revelou que embora a associação tenha uma contribuição devido a interações eletrostáticas, há um caráter de estabilização por interações de curta distância, ligações de H e interações de Van der Waals. Sendo A72 e E96, que estão localizados no motivo KOW, e lisinas (K31, K34, K35, K36, K75 e K125) são importantes para a formação e estabilização do complexo. Em conclusão, o presente trabalho apresenta um conjunto de resultados estruturais e de interação com ácido nucleico para o dSH3thKIN que podem contribuir para um melhor entendimento do papel da proteína KIN nos processos de replicação, transcrição e tradução de DNA.
The KIN protein acts as a DNA damage regulator and participates in the replication of this nucleic acid. Its gene, located on chromosome 10 in human cells, is phylogenetically conserved and ubiquitously expressed in eukaryotes. In humans, this protein has 45 kDa and is structurally composed of four domains: C2H2-type zinc finger domain, central domain homologous to the E. coli RecA protein, nuclear localization signal domain, and SH3-like in tandem domain (dSH3thKIN), made up of two SH3 subdomains. Studies have shown that dSH3thKIN presents in its structure an RNA-binding motif known as KOW motif and, therefore, this domain is considered as the main mediator of interaction of the KIN protein with RNA. This dSH3thKIN/RNA interaction is critical to the performance of KIN's biological activities. However, there is a paucity of structural and interactional information on dSH3thKIN in the literature. Thus, the present work aimed to assign the 1H, 15N and 13C resonances of the domain, determine its three-dimensional structure in solution, characterize the thermal, chemical and pH stability of this domain and investigate its interaction with yeast RNA and homopolymers of RNA and DNA. A total of 98.4% of domain resonances were assigned and the resolution of its three-dimensional structure showed two subdomains with β-barrel arrangements formed by five β-strands each. The analysis of thermal movements of the main chain of dSH3thKIN showed that this domain has reduced internal flexibility despite being formed by two subdomains. The characterization of the thermal and pH stability of the domain revealed an unfolding temperature of around 59 oC, with an aggregation tendency that leads to the formation of structures such as amyloid, and a stability of secondary structures between pH 5.5 and 9.5. The chemical unfolding process of dSH3thKIN occurred reversibly, suggesting that this domain presents an independent folding in the context of the KIN protein. Domain interaction measurements with nucleic acids showed the following affinity order: poly(rG) > poly(rC) > yeast RNA > poly(rU) > poly(rA) > poly(A) DNA, indicating that dSH3thKIN shows a higher affinity for guanine and cytosine bases and yeast RNA. Analysis of the thermodynamic parameters of dSH3thKIN/RNA binding suggests that the major contribution to complex formation is electrostatic interaction. Structural details about the nucleic acid binding site on dSH3thKIN revealed that although the association is contributed due to electrostatic interactions, there is a stabilization character by short distance interactions, H bonds and Van der Waals interactions. Being A72 and E96, which are in the KOW motif, and lysines (K31, K34, K35, K36, K75, and K125) are important for the formation and stabilization of the complex. In conclusion, the present work presents a set of structural and nucleic acid interaction results for dSH3thKIN that may contribute to a better understanding of the role of the KIN protein in DNA replication, transcription, and translation processes.
The KIN protein acts as a DNA damage regulator and participates in the replication of this nucleic acid. Its gene, located on chromosome 10 in human cells, is phylogenetically conserved and ubiquitously expressed in eukaryotes. In humans, this protein has 45 kDa and is structurally composed of four domains: C2H2-type zinc finger domain, central domain homologous to the E. coli RecA protein, nuclear localization signal domain, and SH3-like in tandem domain (dSH3thKIN), made up of two SH3 subdomains. Studies have shown that dSH3thKIN presents in its structure an RNA-binding motif known as KOW motif and, therefore, this domain is considered as the main mediator of interaction of the KIN protein with RNA. This dSH3thKIN/RNA interaction is critical to the performance of KIN's biological activities. However, there is a paucity of structural and interactional information on dSH3thKIN in the literature. Thus, the present work aimed to assign the 1H, 15N and 13C resonances of the domain, determine its three-dimensional structure in solution, characterize the thermal, chemical and pH stability of this domain and investigate its interaction with yeast RNA and homopolymers of RNA and DNA. A total of 98.4% of domain resonances were assigned and the resolution of its three-dimensional structure showed two subdomains with β-barrel arrangements formed by five β-strands each. The analysis of thermal movements of the main chain of dSH3thKIN showed that this domain has reduced internal flexibility despite being formed by two subdomains. The characterization of the thermal and pH stability of the domain revealed an unfolding temperature of around 59 oC, with an aggregation tendency that leads to the formation of structures such as amyloid, and a stability of secondary structures between pH 5.5 and 9.5. The chemical unfolding process of dSH3thKIN occurred reversibly, suggesting that this domain presents an independent folding in the context of the KIN protein. Domain interaction measurements with nucleic acids showed the following affinity order: poly(rG) > poly(rC) > yeast RNA > poly(rU) > poly(rA) > poly(A) DNA, indicating that dSH3thKIN shows a higher affinity for guanine and cytosine bases and yeast RNA. Analysis of the thermodynamic parameters of dSH3thKIN/RNA binding suggests that the major contribution to complex formation is electrostatic interaction. Structural details about the nucleic acid binding site on dSH3thKIN revealed that although the association is contributed due to electrostatic interactions, there is a stabilization character by short distance interactions, H bonds and Van der Waals interactions. Being A72 and E96, which are in the KOW motif, and lysines (K31, K34, K35, K36, K75, and K125) are important for the formation and stabilization of the complex. In conclusion, the present work presents a set of structural and nucleic acid interaction results for dSH3thKIN that may contribute to a better understanding of the role of the KIN protein in DNA replication, transcription, and translation processes.
Descrição
Palavras-chave
Domínio SH3, Ligação tandem, KIN humana, Interação proteína-ácido nucleico, Reparo DNA, SH3 domain, Tandem, Human KIN, Protein-nucleic acid interaction, DNA repair, RMN, NMR