Integrin α1 Has a Long Helix, Extending from the Transmembrane Region to the Cytoplasmic Tail in Detergent Micelles

<div><p>Integrin proteins are very important adhesion receptors that mediate cell-cell and cell-extracellular matrix interactions. They play essential roles in cell signaling and the regulation of cellular shape, motility, and the cell cycle. Here, the transmembrane and cytoplasmic (TMC) domains of integrin α1 and β1 were over-expressed and purified in detergent micelles. The structure and backbone relaxations of α1-TMC in LDAO micelles were determined and analyzed using solution NMR. A long helix, extending from the transmembrane region to the cytoplasmic tail, was observed in α1-TMC. Structural comparisons of α1-TMC with reported αIIb-TMC domains indicated different conformations in the transmembrane regions and cytoplasmic tails. An NMR titration experiment indicated weak interactions between α1-TMC and β1-TMC through several α1-TMC residues located at its N-terminal juxta-transmembrane region and C-terminal extended helix region.</p></div

Structural comparison of integrin α1 and αIIb.

<p>Structure of α1-TMC (1135–1179) and backbone structure comparisons of α1-TM(1142–1169) with αIIb-TM structures indicate different bent regions in the transmembrane helix. The PDB number for each structure is listed below. (A) Structure ensemble of integrin α1-TMC in LDAO micelles; (B) Structure ensemble of integrin α1-TM in LDAO; (C) αIIb-TM (966–993) in bicelles <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062954#pone.0062954-Lau3" target="_blank">[22]</a>; (D) αIIb-TM from IntαIIb/β3 complex in bicelles <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062954#pone.0062954-Lau1" target="_blank">[20]</a>; (E) αIIb-TM (966–993) from αIIb/β3 complex in organic/aqueous solvents, 50% CD₃CN/50% H₂O <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062954#pone.0062954-Yang1" target="_blank">[19]</a>.</p

Resonance assignment and Backbone ¹⁵N relaxation analysis of integrin α1-TMC in LDAO micelles.

<p>(A) Resonance assignment of integrin α1-TMC in LDAO micelles. Site-specific analysis of backbone amide ¹⁵N longitudinal relaxation T1 (B), transverse relaxation T2 (C) and steady-state ¹H-¹⁵N NOE (D) of integrin α1-TMC in LDAO micelles.</p

Solution NMR structure of human integrin α1-TMC in LDAO micelles.

<p>(A) The backbone superposition of the final ten structures with the lowest energies. (B) Cartoon representation of the structure of integrin α1-TMC. G1152 indicates the position of the transmembrane helix kink.</p

Application of Site-Specific ¹⁹F Paramagnetic Relaxation Enhancement to Distinguish two Different Conformations of a Multidomain Protein

Paramagnetic relaxation enhancement (PRE) provides long-distance restraints in solution NMR protein structural studies. It has been shown previously that L27tan, a protein with tandem L27-domains, has two possible conformations. Here, ¹⁹F was site-specifically introduced to L27tan via the incorporation of an unnatural amino acid, trifluoromethyl-phenylalanine (tfmF). Different ¹⁹F signal intensity attenuations were observed at different L27tan sites, due to different distances between the site-specifically incorporated tfmF and site-directed spin radical labeling. Analysis of the ¹⁹F detection PRE showed that the L27tan protein had a closed conformation in solution. This ¹⁹F detection PRE method could be further applied in distance measurements for proteins of large size, including multidomain proteins or membrane proteins

Solution NMR of MPS-1 Reveals a Random Coil Cytosolic Domain Structure

<div><p><i>Caenorhabditis elegans</i> MPS1 is a single transmembrane helical auxiliary subunit that co-localizes with the voltage-gated potassium channel KVS1 in the nematode nervous system. MPS-1 shares high homology with KCNE (potassium voltage-gated channel subfamily E member) auxiliary subunits, and its cytosolic domain was reported to have a serine/threonine kinase activity that modulates KVS1 channel function via phosphorylation. In this study, NMR spectroscopy indicated that the full length and truncated MPS-1 cytosolic domain (134–256) in the presence or absence of n-dodecylphosphocholine detergent micelles adopted a highly flexible random coil secondary structure. In contrast, protein kinases usually adopt a stable folded conformation in order to implement substrate recognition and phosphoryl transfer. The highly flexible random coil secondary structure suggests that MPS-1 in the free state is unstructured but may require a substrate or binding partner to adopt stable structure required for serine/threonine kinase activity.</p></div

Schematic diagram of full length and truncated MPS-1.

<p>(A) Various MPS-1 constructs with MPS-1(1–256), MPS-1(74–256) and MPS-1(134–256) depicted from top to bottom. The black segment (residues 46 to 68) represents the predicted transmembrane helix; (B) Model of the MPS1 domains showing the two phosphorylation sites within the cytoplasmic C-terminal domain. (C) SDS-PAGE of the purified MPS-1 proteins. Lane 1: molecular weight marker; lanes 2-4: purified MPS-1(1–256), MPS-1(74–256) and MPS-1(134–256).</p

Two-dimensional ¹H-¹⁵N HSQC spectra of ¹⁵N labeled full-length and truncated MPS-1 under different buffer conditions at 25°C.

<p>(A) Full-length MPS-1(1–256) spectrum in DPC micelles; (B) MPS-1(134–256) spectrum in DPC micelles; (C) MPS-1(74–256) spectrum in aqueous buffer; (D) MPS-1(134–256) spectrum in aqueous buffer.</p

Secondary structure and backbone relaxation analysis of MPS-1(134–256) in the absence or presence of DPC micelles.

<p>(A) TALOS + secondary structure prediction were based on the assigned backbone ¹³CO, ¹³C_α, ¹³C_β chemical shifts of MPS-1(134–256) in the presence or absence of DPC. Only random coil secondary structure was observed for MPS-1(134–256) in both aqueous buffer and DPC micelles. (See Fig. S3 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111035#pone.0111035.s001" target="_blank">file S1</a> for the Y-axis scale from 0 to 0.3) (B) Distribution of the measured ¹⁵N T₁ longitudinal and T₂ transverse relaxation times and steady-state ¹H-¹⁵N NOE values along the primary sequence in the absence (black) or presence (red) of DPC micelles.</p

¹H-¹⁵N HSQC spectra showing the crosspeak assignments of MPS-1(134–256) in the absence or presence of DPC micelles.

<p>(A) Identifies of a total of 90 of 111 non-proline amino acids were based on (N)H, N, C_α, C_β and CO correlations were made for MPS-1(134–256); (B) In contrast, 66 amino acids were assigned for for MPS-1(134–256) in DPC micelles.</p