IRBIT activates NBCe1-B by releasing the auto-inhibition module from the transmembrane domain.
IRBIT activates NBCe1-B by releasing the auto-inhibition module from the transmembrane domain.
复制标题
IRBIT 通过从跨膜域释放自动抑制模块来激活 NBCe1-B
DOI：
10.1113/jp280578
复制
发表时间：
2021-03
期刊：
影响因子：
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通讯作者：
Chen LM
中科院分区：
文献类型：
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作者：
Su P;Wu H;Wang M;Cai L;Liu Y;Chen LM
生电型Na⁺/HCO₃⁻协同转运蛋白NBCe1 - B在许多组织中广泛表达，包括胰腺、下颌下腺、脑、心脏等。由于自身抑制作用，NBCe1 - B在基础条件下活性很低，但可通过与1,4,5 - 三磷酸肌醇（IRBIT）释放的IP3R结合蛋白相互作用而被完全激活。
在本研究的系统突变以及以往研究的数据基础上，对NBCe1 - B的自身抑制结构域和IRBIT结合结构域的结构成分进行了精细的表征。
减少跨膜结构域胞质侧的负电荷可大大降低NBCe1 - B自身抑制的程度。
我们提出，自身抑制结构域作为一个制动模块发挥作用，通过静电吸引与跨膜结构域结合使NBCe1 - B失活；IRBIT通过与自身抑制结构域竞争性结合，将制动从跨膜结构域释放，从而激活NBCe1 - B。
生电型Na⁺/HCO₃⁻协同转运蛋白NBCe1 - B在体内许多组织中广泛表达。由于其独特的氨基末端的自身抑制结构域（AID）的作用，NBCe1 - B仅表现出基础活性。然而，NBCe1 - B可通过与1,4,5 - 三磷酸肌醇（IRBIT）释放的IP3R结合蛋白相互作用而被激活。在此，我们研究了NBCe1 - B自身抑制及其被IRBIT激活的分子机制。NBCe1 - B的IRBIT结合结构域（IBD）跨越第1 - 52位氨基酸残基，主要由两个臂组成，一个带负电荷（第1 - 24位残基），另一个带正电荷（第40 - 52位残基）。AID主要跨越第40 - 85位残基，在带正电荷的臂上与IBD重叠。通过改变AID中带正电荷的残基，或者将跨膜结构域中的一组带负电荷的残基替换为中性残基，可大大降低NBCe1 - B自身抑制的程度。通过将IRBIT的PEST结构域中的一组带负电荷的天冬氨酸/谷氨酸残基（突变为天冬酰胺/谷氨酰胺）以及一组丝氨酸/苏氨酸残基（突变为丙氨酸），可消除IRBIT与NBCe1 - B之间的相互作用。然而，将PEST结构域中的同一组丝氨酸/苏氨酸残基替换为天冬氨酸并不影响这种相互作用。我们提出：（1）AID作为一个制动器，通过静电相互作用与跨膜结构域结合，使NBCe1 - B减速；（2）IRBIT通过将制动器从跨膜结构域释放来激活NBCe1 - B。
生电型Na⁺/HCO₃⁻协同转运蛋白NBCe1 - B在许多组织中广泛表达，包括胰腺、下颌下腺、脑、心脏等。由于自身抑制作用，NBCe1 - B在基础条件下活性很低，但可通过与1,4,5 - 三磷酸肌醇（IRBIT）释放的IP3R结合蛋白相互作用而被完全激活。
在本研究的系统突变以及以往研究的数据基础上，对NBCe1 - B的自身抑制结构域和IRBIT结合结构域的结构成分进行了精细的表征。
减少跨膜结构域胞质侧的负电荷可大大降低NBCe1 - B自身抑制的程度。
我们提出，自身抑制结构域作为一个制动模块发挥作用，通过静电吸引与跨膜结构域结合使NBCe1 - B失活；IRBIT通过与自身抑制结构域竞争性结合，将制动从跨膜结构域释放，从而激活NBCe1 - B。
The electrogenic Na+/HCO3 −cotransporter NBCe1‐B is widely expressed in many tissues, including pancreas, submandibular gland, brain, heart, etc. NBCe1‐B has very low activity under basal condition due to auto‐inhibition, but can be fully activated by protein interaction with the IP3R‐binding protein released with inositol 1,4,5‐trisphosphate (IRBIT). The structural components of the auto‐inhibition domain and the IRBIT‐binding domain of NBCe1‐B are finely characterized based on systematic mutations in the present study and data from previous studies. Reducing negative charges on the cytosol side of the transmembrane domain greatly decreases the magnitude of the auto‐inhibition of NBCe1‐B. We propose that the auto‐inhibition domain functions as a brake module that inactivates NBCe1‐B by binding to, via electrostatic attraction, the transmembrane domain; IRBIT activates NBCe1‐B by releasing the brake from the transmembrane domain via competitive binding to the auto‐inhibition domain. The electrogenic Na+/HCO3 − cotransporter NBCe1‐B is widely expressed in many tissues in the body. NBCe1‐B exhibits only basal activity due to the action of the auto‐inhibition domain (AID) in its unique amino‐terminus. However, NBCe1‐B can be activated by interaction with the IP3R‐binding protein released with inositol 1,4,5‐trisphosphate (IRBIT). Here, we investigate the molecular mechanism underlying the auto‐inhibition of NBCe1‐B and its activation by IRBIT. The IRBIT‐binding domain (IBD) of NBCe1‐B spans residues 1−52, essentially consisting of two arms, one negatively charged (residues 1−24) and the other positively charged (residues 40−52). The AID mainly spans residues 40−85, overlapping with the IBD in the positively charged arm. The magnitude of auto‐inhibition of NBCe1‐B is greatly decreased by manipulating the positively charged residues in the AID or by replacing a set of negatively charged residues with neutral ones in the transmembrane domain. The interaction between IRBIT and NBCe1‐B is abolished by mutating a set of negatively charged Asp/Glu residues (to Asn/Gln) plus a set of Ser/Thr residues (to Ala) in the PEST domain of IRBIT. However, this interaction is not affected by replacing the same set of Ser/Thr residues in the PEST domain with Asp. We propose that: (1) the AID, acting as a brake, binds to the transmembrane domain via electrostatic interaction to slow down NBCe1‐B; (2) IRBIT activates NBCe1‐B by releasing the brake from the transmembrane domain. The electrogenic Na+/HCO3 −cotransporter NBCe1‐B is widely expressed in many tissues, including pancreas, submandibular gland, brain, heart, etc. NBCe1‐B has very low activity under basal condition due to auto‐inhibition, but can be fully activated by protein interaction with the IP3R‐binding protein released with inositol 1,4,5‐trisphosphate (IRBIT). The structural components of the auto‐inhibition domain and the IRBIT‐binding domain of NBCe1‐B are finely characterized based on systematic mutations in the present study and data from previous studies. Reducing negative charges on the cytosol side of the transmembrane domain greatly decreases the magnitude of the auto‐inhibition of NBCe1‐B. We propose that the auto‐inhibition domain functions as a brake module that inactivates NBCe1‐B by binding to, via electrostatic attraction, the transmembrane domain; IRBIT activates NBCe1‐B by releasing the brake from the transmembrane domain via competitive binding to the auto‐inhibition domain.