Quantitative approaches to utilizing mutational analysis and disulfide crosslinking for modeling a transmembrane domain

Abstract

The transmembrane domain of chemoreceptor Trg from Escherichia coli contains four transmembrane segments in its native homodimer, two from each subunit. We had previously used mutational analysis and sulfhydryl crosslinking between introduced cysteines to obtain data relevant to the three-dimensional organization of this domain. In the current study we used Fourier analysis to assess these data quantitatively for periodicity along the sequences of the segments. The analyses provided a strong indication of a-helical periodicity in the first transmembrane segment and a substantial indication of that periodicity for the second segment. On this basis, we considered both segments as idealized a-helices and proceeded to model the transmembrane domain as a unit of four helices. For this modeling, we calculated helical crosslinking moments, parameters analogous to helical hydrophobic moments, as a quantitative way of condensing and utilizing a large body of crosslinking data. Crosslinking moments were used to define the relative separation and orientation of helical pairs, thus creating a quantitatively derived model for the transmembrane domain of Trg. Utilization of Fourier transforms to provide a quantitative indication of periodicity in data from analyses of transmembrane segments, in combination with helical crosslinking moments to position helical pairs should be useful in modeling other transmembrane domains.

Ala‐insertion scanning mutagenesis of the glycophorin a transmembrane helix: A rapid way to map helix‐helix interactions in integral membrane proteins

“…Cys-scanning of the Lacy protein has so far identified only a handful of functionally critical residues of more than 300 that have been mutated (Kaback et al, 1994); in the Trg chemoreceptor transmembrane segments, only one of 54 Cys mutants was found to be functionally inactive (Lee & Hazelbauer, 1995); and random mutagenesis of the diacylglycerol kinase protein uncovered only a handful of functionally critical residues of more than 120 mutated residues (Wen et al, 1996). In general, integral membrane proteins thus appear to be very resilient to replacement mutagenesis, and we suggest that an Ala-insertion scan followed by saturation mutagenesis may be a more efficient strategy for mapping helix-helix interactions.…”

Structure of a conserved receptor domain that regulates kinase activity: the cytoplasmic domain of bacterial taxis receptors

“…All of the MTP receptors for which structural information exists are family A chemoreceptors from the homologous chemotaxis pathways of E. coli and S. typhimurium [1••,2••, 4,7,[15][16][17][18][19][20][21][22][23][24][25][26][27]. These chemoreceptors are homodimers and possess a large periplasmic domain formed by the association of two identical, antiparallel four-helix bundles, one from each subunit [15][16][17][18][19].…”

“…These chemoreceptors are homodimers and possess a large periplasmic domain formed by the association of two identical, antiparallel four-helix bundles, one from each subunit [15][16][17][18][19]. The transmembrane region is a single antiparallel four-helix bundle formed by the pairing of two membrane-spanning α helices from each subunit [20][21][22][23][24][25][26][27]. Thus, the periplasmic and transmembrane architectures of selected family A chemoreceptors are well understood and are likely to be representative of many family A members.…”

“…The primary advantages of the chemical approach, which employs site-directed sulfhydryl chemistry and spectroscopy to map out secondary structure and tertiary contacts, include the ability to probe structure in the fulllength, membrane-bound protein in vitro or even in vivo [20][21][22][23][24][25][26][27][47][48][49][50]. This approach begins with a cysteine scanning and solvent exposure analysis of the targeted region, which, as described above, has identified extensive helical regions within the cytoplasmic domain.…”

Mutational analysis of a transmembrane segment in a bacterial chemoreceptor

“…The longer helices form a quasi-four-helix bundle along the subunit interface that passes through the membrane, with ␣1 extending from transmembrane helix 1 (TM1) and ␣4 becoming TM2. Cross-linking studies indicate that there are extensive interactions between TM1 and TM2 within a subunit and between TM1 and TM1Ј across the subunit interface (16,18,29), in a pattern that suggests a splayed bundle of the four transmembrane helices.Transmembrane signaling by chemoreceptors is thought to involve conformational change within a stable homodimer (23). What is the nature of that conformational change?…”