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. 2014 Dec 19;289(51):35111-23.
doi: 10.1074/jbc.M114.609446. Epub 2014 Oct 22.

Structure of Transmembrane Domain of Lysosome-Associated Membrane Protein Type 2a (LAMP-2A) Reveals Key Features for Substrate Specificity in Chaperone-Mediated Autophagy

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Structure of Transmembrane Domain of Lysosome-Associated Membrane Protein Type 2a (LAMP-2A) Reveals Key Features for Substrate Specificity in Chaperone-Mediated Autophagy

Ashok K Rout et al. J Biol Chem. .
Free PMC article

Abstract

Chaperone-mediated autophagy (CMA) is a highly regulated cellular process that mediates the degradation of a selective subset of cytosolic proteins in lysosomes. Increasing CMA activity is one way for a cell to respond to stress, and it leads to enhanced turnover of non-critical cytosolic proteins into sources of energy or clearance of unwanted or damaged proteins from the cytosol. The lysosome-associated membrane protein type 2a (LAMP-2A) together with a complex of chaperones and co-chaperones are key regulators of CMA. LAMP-2A is a transmembrane protein component for protein translocation to the lysosome. Here we present a study of the structure and dynamics of the transmembrane domain of human LAMP-2A in n-dodecylphosphocholine micelles by nuclear magnetic resonance (NMR). We showed that LAMP-2A exists as a homotrimer in which the membrane-spanning helices wrap around each other to form a parallel coiled coil conformation, whereas its cytosolic tail is flexible and exposed to the cytosol. This cytosolic tail of LAMP-2A interacts with chaperone Hsc70 and a CMA substrate RNase A with comparable affinity but not with Hsp40 and RNase S peptide. Because the substrates and the chaperone complex can bind at the same time, thus creating a bimodal interaction, we propose that substrate recognition by chaperones and targeting to the lysosomal membrane by LAMP-2A are coupled. This can increase substrate affinity and specificity as well as prevent substrate aggregation, assist in the unfolding of the substrate, and promote the formation of the higher order complex of LAMP-2A required for translocation.

Keywords: Autophagy; CMA; Chaperone; DPC; LAMP-2; Micelles; Nuclear Magnetic Resonance (NMR); Protein Structure; Transport.

Figures

FIGURE 1.
FIGURE 1.
A, the two-dimensional 15N,1H HSQC spectrum of TM domain of human LAMP-2A in DPC micelles. The assignments are indicated by the one-letter amino acid code followed by the corresponding number along the protein primary sequence. The resonances connected with horizontal lines are correlations from side chain NH2 spin pairs belonging to Asn and Gln residues. B, secondary structures of TM domain of LAMP-2A in DPC micelles. The secondary chemical shift index ΔCα − ΔCβ defines the presence of one TM helix. The values of ΔCα and ΔCβ were obtained as the differences between the experimentally observed 13Cα and 13Cβ chemical shifts and the corresponding random coil chemical shifts. The consecutive positive bars in the chemical shift index plot indicate the presence of α-helical conformation for the TM domain, whereas the cytosolic and N-terminal tails do not adopt any specific secondary conformation. The defined TM helix is also identified by the characteristic medium range interproton NOE connectivity of Hαi to Hβi + 3 and strong HNi to HNi + 1 NOE. The secondary structural elements are shown at the bottom of panel B. C, impact of solvent PRE on amide proton transverse relaxation rate (1HN2) of TM domain of LAMP-2A. No significant effect could be observed in 1HN2 for the TM domain, confirming its insertion in the DPC micelles. Residue numbering is kept the same as the full-length LAMP-2A.
FIGURE 2.
FIGURE 2.
Backbone 15N relaxation dynamics of TM domain of human LAMP-2A in DPC micelles. The 15N T1 (A) and T2 (B) relaxation data of TM-LAMP-2A and their ratio (C) are plotted as a function of residue number. Relaxation values could not be estimated for residues Asp373, Ala380, Val381, Ala386, Leu400, and Tyr407 due to spectral overlap and weak intensity resulting in improper fit. The residues Ser369, Ala370, Val377, Pro378, His403, and His404 are not assigned. The mean value of T1 and T2 for the structured region is 1153.5 ± 20.1 and 55.1 ± 2.9 ms, respectively, corresponding to an estimated rotational correlation time (τc) of 14.4 ns. The secondary structural elements are shown at the top of A. Error bars represent fitting errors in the relaxation times.
FIGURE 3.
FIGURE 3.
Sedimentation equilibrium of human TM domain of LAMP-2A in DPC micelles. A, the sedimentation curves obtained at centrifugation speeds of 17,000 (red), 22,000 (blue), and 32,000 (black) rpm (solid circles). These curves were collected with a protein concentration of 365 μm. The sedimentation equilibrium curves acquired with the density matching protocol were best fit to a monomer-trimer equilibrium (global χ2 = 0.34), and the fitting result is shown as a solid line. B, the residuals of the above fitting routine. C, chemical cross-linking of TM-LAMP-2A characterized by SDS-PAGE using a silver staining protocol. The silver-stained SDS-polyacrylamide gel of the reference sample (left panel) and that of TM-LAMP-2A in the presence of chemical cross-linker dimethyl suberimidate (DMS) (right panel) are shown. Lane labeled M contains molecular mass markers.
FIGURE 4.
FIGURE 4.
NMR-derived solution structure of TM domain of human LAMP-2A in DPC micelles. A, a superposition of an ensemble of 20 lowest energy structures of TM-LAMP-2A (backbone root mean square deviation, 0.52 ± 0.14 Å). The calculated structures show an extended α-helical transmembrane domain and a flexible cytosolic tail at the C terminus. B, selected strips of 13C-filtered NOESY at the Val388-Hα and Tyr396-Hα chemical shifts obtained from a sample composed of a mixture of 13C/15N-labeled and unlabeled TM-LAMP-2A in a 1:1 ratio. C, ensemble of 20 superimposed lowest energy structures of TM-LAMP-2A trimer (backbone root mean square deviation, 0.67 ± 0.16 Å). In the calculated structure of the trimer, three TM helices wrap around each other to form a symmetric coiled coil conformation. For clarity, only the structured region of TM domain is shown here. D, lowest energy structure of the trimer showing residues Val381, Leu385, Val388, Leu389, Val392, Ala395, and Tyr396 forming the core of the trimer. In this figure, A, B, and C represents three individual subunits. E, the top view (N terminus) of the TM-LAMP-2A showing some of the hydrophobic interactions that stabilize the core of the trimer.
FIGURE 5.
FIGURE 5.
A, the PRE from mixing the spin probe attached to unlabeled TM-LAMP-2A with 15N-labeled TM-LAMP-2A was determined by measuring the change in relaxation rates, 1HN2, relative to a sample not containing the spin label. The enhanced 1HN2 indicates the tight association of the TM-LAMP-2A monomers in the micelles. B, correlation between 1HN2 and the calculated sum of inversed sixth power distances from individual backbone amide proton from each monomer to the spin label based on the NMR-derived trimer structure of TM-LAMP-2A. Error bars correspond to reproducibilities from two different data sets.
FIGURE 6.
FIGURE 6.
A, overlay of the contour plot of 1H-15N backbone resonances of those residues of TM-LAMP-2A that undergo significant chemical shift changes during chaperone Hsc70 titration (black and red correspond to unbound and bound TM-LAMP-2A, respectively). B, residue-specific CSP of TM domain of LAMP-2A upon addition of the substrate binding domain of Hsc70. The secondary structural elements are shown at the top of B. Residues experiencing significant perturbations are those with CSP values above the standard of deviation (0.016) cutoff shown in a dotted line. These residues belong to the cytosolic tail of LAMP-2A. C, a ribbon representation of TM-LAMP-2A with residues having significant chemical shift perturbation shown as stick models. D and E, fitting of the experimental chemical shift titration data (circle, Lys401; diamond, His402; triangle, Ala405; star, Gly406) to determine Kd for LAMP-2A binding to Hsc70 and RNase A, respectively. The results from the fit are shown as solid lines.

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