RESUMEN
The band 3 transporter is a critical integral membrane protein of the red blood cell (RBC), as it is responsible for catalyzing the exchange of bicarbonate and chloride anions across the plasma membrane. To elucidate the structural mechanism of the band 3 transporter, detergent solubilized human ghost membrane reconstituted in nanodiscs was applied to a cryo-EM holey carbon grid to define its composition. With this approach, we identified and determined structural information of the human band 3 transporter. Here, we present 5 different cryo-EM structures of the transmembrane domain of dimeric band 3, either alone or bound with chloride or bicarbonate. Interestingly, we observed that human band 3 can form both symmetric and asymmetric dimers with a different combination of outward-facing (OF) and inward-facing (IF) states. These structures also allow us to obtain the first model of a human band 3 molecule at the IF conformation. Based on the structural data of these dimers, we propose a model of ion transport that is in favor of the elevator-type mechanism.
Asunto(s)
Proteína 1 de Intercambio de Anión de Eritrocito , Bicarbonatos , Cloruros , Microscopía por Crioelectrón , Humanos , Microscopía por Crioelectrón/métodos , Bicarbonatos/metabolismo , Cloruros/metabolismo , Proteína 1 de Intercambio de Anión de Eritrocito/metabolismo , Proteína 1 de Intercambio de Anión de Eritrocito/química , Transporte Iónico , Modelos Moleculares , Multimerización de Proteína , Conformación Proteica , Membrana Celular/metabolismoRESUMEN
Band 3, or anion exchanger 1 (AE1), is one of the indispensable transmembrane proteins involved in the effective respiratory process of the human body and is primarily responsible for the exchange of bicarbonate and chloride anions across the plasma membrane of erythrocyte. However, the molecular mechanism of ion transport of Band 3 is not completely understood, yet. In this work, we systematically investigate the key binding sites of bicarbonate ions in Band 3 and the impact of cholesterol (CHOL) in lipid bilayers on bicarbonate ion binding using all-atom molecular dynamics (MD) simulations. We examine the dynamics of interactions of bicarbonate ions with Band 3 in the microsecond time scale and calculate the binding free energy of the anion in Band 3. The results indicate that the residue R730 of Band 3 is the most probable binding site for bicarbonate ions. CHOL enhances the bicarbonate ion binding by influencing the conformational stability of Band 3 and compressing the volume of the Band 3 cavity. These findings provide some insights into the bicarbonate ion binding in Band 3 and are helpful for understanding the anion exchange of Band 3.
Asunto(s)
Proteína 1 de Intercambio de Anión de Eritrocito , Bicarbonatos , Colesterol , Simulación de Dinámica Molecular , Proteína 1 de Intercambio de Anión de Eritrocito/química , Proteína 1 de Intercambio de Anión de Eritrocito/metabolismo , Bicarbonatos/química , Bicarbonatos/metabolismo , Colesterol/química , Colesterol/metabolismo , Humanos , Sitios de Unión , Unión Proteica , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismoRESUMEN
Se estandarizó un método inmunoenzimático (ELISA) para la determinación de anticuerpos naturales anti banda 3 acoplando a placas Chemobond la proteína banda 3 obtenida a partir de la membrana de eritrocitos humanos mediante una combinación de cromatografía de intercambio aniónico y afinidad. Se emplearon 3 mg de la proteína banda 3 por pozo, lográndose un nivel de detección de 1 mg/mL de anticuerpo anti banda 3. Los resultados indican la utilidad del método para su empleo en la medición de los niveles de anticuerpos naturales anti banda 3 en pacientes con drepanocitosis, tanto en estado basal como en las crisis vasooclusivas dolorosas.
An immunoenzimatic assay (ELISA) was standardized to determine the natural anti-band 3 antibodies by coupling with Chemobond plates the band 3 protein obtained from the membrane of human erythrocytes by a combination of affinity and anion exchange chromatography. 3 g of band 3 protein per well were used. A level of detection of 1 g/mL of anti-band 3 antibody was achieved. The results showed the usefulness of the method to measure the levels of natural anti-band 3 antibodies in patients with drepanocytes, both in basal state and in the painful vasocclusive crises.