The ribose high-affinity transporter of Escherichia coli is one of the binding protein dependent ABC transporters. It is composed of the periplasmic ribose-binding protein (RBP), the hydrophobic membrane protein (RbsC), and ATP-binding protein (RbsA). To investigate interaction between RBP and permease, regions on the tertiary structure of RBP that interact with the membrane permease were genetically probed by screening a mutation using the chimeric receptor Trz. The mutational changes were repeatedly found in several residues of RBP, concentrating on three surface regions and comprising two domains of the tertiary structure. The two regions, including residues 52 and 166, are specifically involved in the permease interaction while the third region including residues 72, 134, and some others recognizes both the permease and the chemosensory receptor. To understand the structure and function of RbsC, topology of RbsC was investigated by an alkaline phosphatase fusion. Characterization of 64 RbsC-PhoA fusions, isolated either specifically or randomly, revealed that the RbsC protein is composed of six transmembrane helices with three periplasmic loops. In order to confirm the cytoplasmic location of the short C-terminal region (5 a.a.), inside-out or right side-out membrane vesicles were generated in which only in inside-out vesicles the C-terminal region was found to be digested by carboxypeptidase A. This result is consistent with the model, based on the results of alkaline phosphatase fusions, that the protein traverses membrane six times and the N- and C-termini being exposed to the cytoplasm. For the RBP mutations previously characterized (Eym et al., 1996), permease suppressors were isolated in RbsC after a series of enrichments. Based on RbsC topology which contains six transmembrane regions (Park and Park, submitted), suppressors of RbsC for mutations of N- and C-domains of RBP were localized into the last transmembrane region and the cytoplasmic loop I adjacent to transmembrane III, respectively, suggesting that two domains of RBP interact with different regions of RbsC. In order to examine the possibility of RbsC interacting with RBP as a homodimer, we made combinations between the mutants of RBP each having a mutation in either N- or C-domain and the mutations of RbsC contained in the chimeric dimer tandemly fused between N- and C-termini. Our data indicate that the periplasmic ribose binding protein symmetrically interacts with homodimer of integral membrane protein RbsC, suggesting that the integral membrane proteins of AraH/RbsC super family might function as a homodimer.
대장균의 리보스 high-affinity systme은 미생물의 결합 단백질에 의존하는 ABC 수송체의 일종이다. 이 수송체는 리보스 결합 단백질 (RBP), 막 단백질 (RbsC), ATP 결합 단백질 (RbsA)로 이루어져 있다. 우리는, 리보스 결합 단백질과 투과효소와의 상호 작용을 이해하기 위해서, 먼저리보스 결합 단백질의 상호 적용부의르 알아냈다. 리보스 결합 단백질의 N-domain의 D52와 G72 sites; C-domain의 G134와 R166 sites가 투과효소와의 상호 작용부위이다. 투과효소의 어느 성분이 리보스 결합 단백질과 상호 작용하는 지를 알기 위해서, 위의 돌연변이들을 이용하였다. 리보스 성장을 회복하는 돌연변이를 얻어서 분석해보니까, 상호 작용에 관련된 부위는 모두 막 단백질 (RbsC)에 나타났다. 이 회복 돌연변이의 위치를 알기 위해서, 막 단백질 (RbsC)의 구조를 분석하였다. RbsC는 6개의 transmembrane helices를 가지고 있고, RbsC의 N-과 C-termini는 모두 cytoplasm에 존재한다. 리보스 결합 단백질의 돌연변이와 막 단백질 (RbsC)의 회복 돌연변이의 조합으로써, 리보스 결합 단백질 (RBP)가 막 단백질 (RbsC)두 개와 대칭적으로 상호 작용한다는 것을 알아냈다.
