Structure and mechanisms of transport of human Asc1/CD98hc amino acid transporter

Recent cryoEM studies elucidated details of the structural basis for the substrate selectivity and translocation of heteromeric amino acid transporters. However, Asc1/CD98hc is the only neutral heteromeric amino acid transporter that can function through facilitated diffusion, and the only one that efficiently transports glycine and D-serine, and thus has a regulatory role in the central nervous system. Here we use cryoEM, ligand-binding simulations, mutagenesis, transport assays, and molecular dynamics to define human Asc1/CD98hc determinants for substrate specificity and gain insights into the mechanisms that govern substrate translocation by exchange and facilitated diffusion. The cryoEM structure of Asc1/CD98hc is determined at 3.4-3.8 angstrom resolution, revealing an inward-facing semi-occluded conformation. We find that Ser 246 and Tyr 333 are essential for Asc1/CD98hc substrate selectivity and for the exchange and facilitated diffusion modes of transport. Taken together, these results reveal the structural bases for ligand binding and transport features specific to human Asc1. Asc1/CD98hc is a key regulator of small neutral amino acid transport in the brain and adipose tissue. Here, authors report the structure of semi-occluded hAsc1/CD98hc and provide a model for Asc1 exchange and facilitated diffusion modes of transport

Tripeptide binding in a proton-dependent oligopeptide transporter

Proton‐dependent oligopeptide transporters (POTs) are important for the uptake of di‐/tripeptides in many organisms and for drug transport in humans. The binding mode of dipeptides has been well described. However, it is still debated how tripeptides are recognized. Here, we show that tripeptides of the sequence Phe‐Ala‐Xxx bind with similar affinities as dipeptides to the POT transporter from Streptococcus thermophilus (PepT$_{St}$). We furthermore determined a 2.3‐Å structure of PepT$_{St}$ in complex with Phe‐Ala‐Gln. The phenylalanine and alanine residues of the peptide adopt the same positions as previously observed for the Phe‐Ala dipeptide, while the glutamine side chain extends into a hitherto uncharacterized pocket. This pocket is adaptable in size and can likely accommodate a wide variety of peptide side chains

Structure determination of a major facilitator peptide transporter: Inward facing PepT_St from <i>Streptococcus thermophilus</i> crystallized in space group P3₁21

<div><p>Major facilitator superfamily (MFS) peptide transporters (typically referred to as PepT, POT or PTR transporters) mediate the uptake of di- and tripeptides, and so play an important dietary role in many organisms. In recent years, a better understanding of the molecular basis for this process has emerged, which is in large part due to a steep increase in structural information. Yet, the conformational transitions underlying the transport mechanism are still not fully understood, and additional data is therefore needed. Here we report in detail the detergent screening, crystallization, experimental MIRAS phasing, and refinement of the peptide transporter PepT_St from <i>Streptococcus thermophilus</i>. The space group is P3₁21, and the protein is crystallized in a monomeric inward facing form. The binding site is likely to be somewhat occluded, as the lobe encompassing transmembrane helices 10 and 11 is markedly bent towards the central pore of the protein, but the extent of this potential occlusion could not be determined due to disorder at the apex of the lobe. Based on structural comparisons with the seven previously determined P2₁2₁2₁ and C222₁ structures of inward facing PepT_St, the structural flexibility as well as the conformational changes mediating transition between the inward open and inward facing occluded states are discussed. In conclusion, this report improves our understanding of the structure and conformational cycle of PepT_St, and can furthermore serve as a case study, which may aid in supporting future structure determinations of additional MFS transporters or other integral membrane proteins.</p></div

Comparisons of crystal packing.

<p><i>(a)</i> Crystal packing in the P3₁21 form. Top: slice of the crystal taken parallel to the <i>(a-b)</i>-plane and encompassing one layer of molecules. The PepT_St molecules are colored magenta. Bottom: slice of the crystal taken parallel to the <i>(b-c)</i>-plane. <i>(b)</i> Crystal packing in the P2₁2₁2₁ form (PDB: 4APS). Depicted as in panel <i>a</i>, except that the two molecules in the asymmetric unit are colored light and dark blue, and that the dimer formed by these are boxed. <i>(c)</i> Crystal packing in the C222₁ form (PDB: 4D2C). Depicted as in panel <i>a</i>, except that the molecules are colored green, and that a dimer with the same arrangement as seen in the P2₁2₁2₁ form is similarly boxed, though this dimer is generated by crystal symmetry rather than being non-crystallographic. Crystal packing interfaces are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173126#pone.0173126.s001" target="_blank">S1 Fig</a>.</p

Refinement statistics.

<p>Refinement statistics.</p

MIRAS phasing statistics.

<p>MIRAS phasing statistics.</p

Multispecific Substrate Recognition in a Proton-Dependent Oligopeptide Transporter

roton-dependent oligopeptide transporters (POTs) are important for uptake of dietary di- and tripeptides in many organisms, and in humans are also involved in drug absorption. These transporters accept a wide range of substrates, but the structural basis for how different peptide side chains are accommodated has so far remained obscure. Twenty-eight peptides were screened for binding to PepT$_{St}$ from Streptococcus thermophilus, and structures were determined of PepT$_{St}$ in complex with four physicochemically diverse dipeptides, which bind with millimolar affinity: Ala-Leu, Phe-Ala, Ala-Gln, and Asp-Glu. The structures show that PepT$_{St}$ can adapt to different peptide side chains through movement of binding site residues and water molecules, and that a good fit can be further aided by adjustment of the position of the peptide itself. Finally, structures were also determined in complex with adventitiously bound HEPES, polyethylene glycol, and phosphate molecules, which further underline the adaptability of the binding site

Structural comparisons.

<p><i>(a)</i> All previously determined PepT_St structures superimposed on the P3₁21 structure (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173126#pone.0173126.t005" target="_blank">Table 5</a>). The P3₁21 structure is magenta, the P2₁2₁2₁ structure is blue the C222₁ structures with dipeptides are green (dipeptides are shown in sticks), and the C222₁ structures without dipeptides are white. Two views are shown—cytoplasmic (top) and periplasmic (bottom). <i>(b)</i> Superimposition of the N-domains of the same structures used in panel <i>a</i>. <i>(c)</i> Superimposition of the C-domains of the same structures used in panel <i>a</i>. <i>(d)</i> Same as in panel <i>b</i>, except that the C-domains are shown alongside the superimposed N-domains and that the P2₁2₁2₁ structure was omitted. A rotation of the C-domain relative to the N-domain can be recognized for the C222₁ structures with dipeptides (rotation marked with arrows). (e) Same as in panel <i>c</i>, except that only TM10 and TM11 are shown, that the C222₁ structure determined at room temperature is grey, and that the P2₁2₁2₁ structure was omitted. The C222₁ structures with dipeptides, the P3₁21 structure and to some extent the C222₁ structure determined at room temperature, show bending of TM11 (indicated with a dashed arrow) and increased disorder in TM10 (indicated with a dashed oval) relative to the other C222₁ structures determined at cryogenic temperatures.</p

Experimental electron density map.

<p>The structure is shown in magenta, and the density modified MIRAS phased electron density map is shown at 1 σ as a grey wire mesh.</p

Site finding.

<p>The structure is magenta and the methionine side chains are shown in sticks and labeled, green and blue spheres designate AutoSHARP SeMet and Au sites, respectively, and the similarly colored wire meshes designate the model phased SeMet and Au anomalous difference Fourier maps contoured at 3 σ. Two views are shown: cytoplasmic (left) and periplasmic (right).</p