Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2011 Oct;1808(10):2403-12.
doi: 10.1016/j.bbamem.2011.06.018. Epub 2011 Jul 6.

Structural Characterization of AS1-membrane Interactions From a Subset of HAMP Domains

Affiliations
Free PMC article

Structural Characterization of AS1-membrane Interactions From a Subset of HAMP Domains

Sofia Unnerståle et al. Biochim Biophys Acta. .
Free PMC article

Abstract

HAMP domains convert an extracellular sensory input into an intracellular signaling response in a wide variety of membrane-embedded bacterial proteins. These domains are almost invariably found adjacent to the inner leaflet of the cell membrane. We therefore examined the interaction of peptides corresponding to either AS1 or AS2 of four different, well-characterized HAMP domains with several membrane model systems. The proteins included an Archaeoglobus fulgidus protein (Af1503), the Escherichia coli osmosensor EnvZ(Ec), the E. coli nitrate/nitrite sensor NarX(Ec), and the aspartate chemoreceptor of E. coli (Tar(Ec)). Far-UV CD and NMR spectroscopy were used to monitor the induction of secondary structure upon association with neutral or acidic large unilamellar vesicles (LUVs) and bicelles. We observed significant increases in α-helicity within AS1 from NarX(Ec) and Tar(Ec) but not in AS1 from the other proteins. To characterize these interactions further, we determined the solution structure of AS1 from Tar(Ec) associated with acidic bicelles. The bulk of AS1 formed an amphipathic α-helix, whereas the N-terminal control cable, the region between TM2 and AS1, remained unstructured. We observed that the conserved prolyl residue found in AS1 of many membrane-adjacent HAMP domains defined the boundary between the unstructured and helical regions. In addition, two positively charged residues that flank the hydrophobic surface of AS1 are thought to facilitate electrostatic interactions with the membrane. We interpret these results within the context of the helix-interaction model for HAMP signaling and propose roles for AS1-membrane interactions during the membrane assembly and transmembrane communication of HAMP-containing receptors.

Figures

Figure 1
Figure 1
Far-UV CD spectra of the AS1- and AS2-containing peptides in the presence of LUVs. The peptides analyzed are constituents of the HAMP domains from (A) Af1503, (B) EnvZEc, (C) NarXEc, and (D) TarEc. Samples contained a final concentration of 50 μM peptide (AS1p-, AS2p- or a 1:1 mix) in 50 mM sodium phosphate buffer (pH 7.2) (left panel), buffer with an additional 1 mM POPC (center panel), or buffer with an additional 1 mM POPC/POPG 4:1 (right panel).
Figure 2
Figure 2
Estimation of the extent of secondary structure in the presence of LUVs. The far-UV CD spectra were further analyzed with Dicroweb [36], using the CONTIN method [37]. The α-helical content of all 8 peptides in 50 mM sodium phosphate buffer (pH 7.2) (white), buffer with an additional 1 mM POPC (light grey), or buffer with an additional 1 mM POPC/POPG 4:1 (dark grey) are shown.
Figure 3
Figure 3
Effect of increasing ionic strength on peptide-membrane interactions. 150 mM KF was added to samples containing AS1p-NarXEc (left panel) and AS1p-TarEc (right panel) in the presence of 50 mM potassium phosphate buffer (pH 7.2) and either 1 mM POPC or 1 mM POPC/POPG 4:1.
Figure 4
Figure 4
Far-UV CD spectra of the AS1- and AS2-containing peptides in the presence of neutral and acidic phospholipid bicelles. Samples contained a final concentration of 500 μM peptide, 50 mM potassium phosphate (pH 7.2), and 150 mM DMPC/DHPC (q=0.5) (left panel) or 150 mM [DMPC/DMPG 4:1]/DHPC (q=0.5) (right panel).
Figure 5
Figure 5
Estimation of the extent of secondary structure from the far-UV CD spectra in the presence of phospholipid bicelles. The α-helical content was determined for all 8 peptides in 50 mM sodium phosphate buffer pH 7.2 (white), buffer with an additional 150 mM DMPC/DHPC (q=0.5) (light grey), or buffer with an additional 150 mM [DMPC/DMPG 4:1]/DHPC (q=0.5) (dark grey).
Figure 6
Figure 6
Secondary structure of AS1p-TarEc as determined by NMR. (a) Secondary Hα chemical shifts of AS1p-TarEc in the presence of acidic bicelles. (b) A summary of the sequential and medium-range NOE-derived distances from the NOESY spectra.
Figure 7
Figure 7
(a) Ensemble of the 25 structures of AS1p-TarEc in 10% negatively charged phospholipid bicelles with the lowest CYANA target function. The side-chains of Pro6, Ile11, His13, Arg15, Ile17 and Gly19 from the average structure in the ensemble are shown. (b) AS1 of TarEc adopts an amphipathic α-helical structure in the presence of acidic bicelles. The position of the hydrophobic (yellow), postively charged (blue), negatively charged (red), and partially positively charged residues (grayish green) are indicated. The side chains of Lys9 and Arg15 that flank this region are indicated as well.
Figure 8
Figure 8
Modulation of AS1-membrane interactions by displacements of TM2. The helix interaction model proposes that AS1 (blue) is oriented nearly parallel to the inner leaflet of the cell membrane in one conformation (left panel) [6]. This would allow the hydrophobic surface of AS1 to interact with the hydrophobic core of the membrane and the flanking positively charged residues to interact with the negatively charged membrane surface as depicted in Figure 7B. In the alternative conformation, AS1 would not interact with the membrane, but rather with its cognate helical partner, AS2 (yellow) (right panel). Displacements of TM2 are predicted to bias this equilibrium by altering the position of AS1 relative to the inner leaflet of the membrane (right panel). The intrinsic properties, such as length and flexibility, of the control cable (black) connecting TM2 (red) and AS1 are also expected to contribute to the baseline equilibrium between these conformations.

Similar articles

See all similar articles

Cited by 5 articles

Publication types

MeSH terms

LinkOut - more resources

Feedback