Functional characterization of the maltose ATP-binding-cassette transporter of Salmonella typhimurium by means of monoclonal antibodies directed against the MalK subunit.
Functional characterization of the maltose ATP-binding-cassette transporter of Salmonella typhimurium by means of monoclonal antibodies directed against the MalK subunit.
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通过针对 MalK 亚基的单克隆抗体对鼠伤寒沙门氏菌的麦芽糖 ATP 结合盒转运蛋白进行功能表征。
DOI：
10.1046/j.1432-1033.2002.03099.x
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发表时间：
2002
期刊：
影响因子：
--
通讯作者：
E. Schneider
中科院分区：
文献类型：
--
作者：
A. Stein;M. Seifert;R. Volkmer‐Engert;Jörg Siepelmeyer;K. Jahreis;E. Schneider
鼠伤寒沙门氏菌的麦芽糖ATP结合盒转运蛋白由膜结合复合物（MalFGK 2）和周质受体（MalE）组成。除了其在运输中的作用外，该复合物还充当麦芽糖调节的基因表达的阻遏物，并且在诱导物排除过程中受到抑制。这些活动被认为是介导的相互作用的ATP酶亚基，MalK，与转录激活因子，MalT，和非磷酸化酶IIA的葡萄糖磷酸转移酶系统，分别。为了进一步了解对这些功能至关重要的蛋白质区域，我们已经产生了9种MalK特异性单克隆抗体。这些结合四个非重叠线性表位：60-LFig-63（5 B5）、113-RVNQVAEVLQL-123（由4 H12表示）、309-GHETQI-314（2F 9）和352-LFREDGSACR-361（由4 B3表示）。所有mAb识别可溶性MalK和MalFGK 2复合物中的表位，Kd值范围为10-6至10-8 m。ATP降低了单克隆抗体对可溶性MalK的亲和力，表明构象变化使表位不易接近。4 H12和5 B5抑制MalK的ATP酶活性和MalE/麦芽糖刺激的蛋白脂质体的ATP酶活性，而它们的Fab片段没有表现出显著的影响。结果表明，在MalK二聚体和完整的复合物中，螺旋3具有类似的溶剂暴露位置，可能会反对在催化过程中的直接作用。4 B3和2F 9分别在MalT和酶IIAGlc的存在下表现出与MalFGK 2复合物的结合降低，从而为MalK的C-末端结构域是与调节蛋白相互作用的位点提供了第一个直接证据。
The maltose ATP-binding cassette transporter of Salmonella typhimurium is composed of a membrane-associated complex (MalFGK2) and a periplasmic receptor (MalE). In addition to its role in transport, the complex acts as a repressor of maltose-regulated gene expression and is subject to inhibition in the process of inducer exclusion. These activities are thought to be mediated by interactions of the ATPase subunit, MalK, with the transcriptional activator, MalT, and nonphosphorylated enzyme IIA of the glucose phosphotransferase system, respectively. To gain further insight in protein regions that are critical for these functions, we have generated nine MalK-specific monoclonal antibodies. These bind to four nonoverlapping linear epitopes: 60-LFig-63 (5B5), 113-RVNQVAEVLQL-123 (represented by 4H12), 309-GHETQI-314 (2F9) and 352-LFREDGSACR-361 (represented by 4B3). All mAbs recognize their epitopes in soluble MalK and in the MalFGK2 complex with Kd values ranging from 10-6 to 10-8 m. ATP reduced the affinity of the mAbs for soluble MalK, indicating a conformational change that renders the epitopes less accessible. 4H12 and 5B5 inhibit the ATPase activity of MalK and the MalE/maltose-stimulated ATPase activity of proteoliposomes, while their Fab fragments displayed no significant effect. The results suggest a similar solvent-exposed position of helix 3 in the MalK dimer and in the intact complex and might argue against a direct role in the catalytic process. 4B3 and 2F9 exhibit reduced binding to the MalFGK2 complex in the presence of MalT and enzyme IIAGlc, respectively, thereby providing the first direct evidence for the C-terminal domain of MalK being the site of interaction with the regulatory proteins.
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影响因子：
--
作者：
J. Hall;A. Davidson;H. Nikaido
通讯作者：
J. Hall;A. Davidson;H. Nikaido
DOI：
10.1016/s0021-9258(18)31535-7
发表时间：
1991-05
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
A. Davidson;H. Nikaido
通讯作者：
A. Davidson;H. Nikaido
捕获 ATP 结合盒转运蛋白的过渡态：麦芽糖转运协同机制的证据。
DOI：
10.1073/pnas.98.4.1525
发表时间：
2001
影响因子：
11.1
作者：
Chen,J;Sharma,S;Quiocho,FA;Davidson,AL
通讯作者：
Davidson,AL
DOI：
10.1073/pnas.89.6.2360
发表时间：
1992-03-15
影响因子：
11.1
作者：
DAVIDSON, AL;SHUMAN, HA;NIKAIDO, H
通讯作者：
NIKAIDO, H