Sequence and structural analysis of the Asp-box motif and Asp-box beta-propellers; a widespread propeller-type characteristic of the Vps10 domain family and several glycoside hydrolase families

<p>Abstract</p> <p>Background</p> <p>The Asp-box is a short sequence and structure motif that folds as a well-defined β-hairpin. It is present in different folds, but occurs most prominently as repeats in β-propellers. Asp-box β-propellers are known to be characteristically irregular and to occur in many medically important proteins, most of which are glycosidase enzymes, but they are otherwise not well characterized and are only rarely treated as a distinct β-propeller family. We have analyzed the sequence, structure, function and occurrence of the Asp-box and s-Asp-box -a related shorter variant, and provide a comprehensive classification and computational analysis of the Asp-box β-propeller family.</p> <p>Results</p> <p>We find that all conserved residues of the Asp-box support its structure, whereas the residues in variable positions are generally used for other purposes. The Asp-box clearly has a structural role in β-propellers and is highly unlikely to be involved in ligand binding. Sequence analysis of the Asp-box β-propeller family reveals it to be very widespread especially in bacteria and suggests a wide functional range. Disregarding the Asp-boxes, sequence conservation of the propeller blades is very low, but a distinct pattern of residues with specific properties have been identified. Interestingly, Asp-boxes are occasionally found very close to other propeller-associated repeats in extensive mixed-motif stretches, which strongly suggests the existence of a novel class of hybrid β-propellers. Structural analysis reveals that the top and bottom faces of Asp-box β-propellers have striking and consistently different loop properties; the bottom is structurally conserved whereas the top shows great structural variation. Interestingly, only the top face is used for functional purposes in known structures. A structural analysis of the 10-bladed β-propeller fold, which has so far only been observed in the Asp-box family, reveals that the inner strands of the blades are unusually far apart, which explains the surprisingly large diameter of the central tunnel of sortilin.</p> <p>Conclusion</p> <p>We have provided new insight into the structure and function of the Asp-box motif and of Asp-box β-propellers, and expect that the classification and analysis presented here will prove helpful in interpreting future data on Asp-box proteins in general and on Asp-box β-propellers in particular.</p

High-resolution insights into binding of unfolded polypeptides by the PPIase chaperone SlpA

SlpA is a 2-domain protein consisting of an FK506-binding protein (FKBP) domain that harbors the peptidyl-prolyl cis/trans-isomerase (PPIase) active site and a small insert-in-flap (IF) domain that endows the protein with chaperone activity. We have determined the structure of SlpA from Escherichia coli at 1.35-Å resolution. The overall structure is similar to other known structures of the FKBP-IF subfamily. However, by serendipity, the linker region of the purification tag binds in the chaperone binding groove of the IF domain, making this the first structure of an FKBP-IF protein in complex with a mimic of an unfolded chaperone substrate. The linker binds by β-sheet augmentation, thus completing the incomplete β barrel of the IF domain and shielding a considerable hydrophobic surface area from the solvent. Interestingly, a proline residue in trans configuration appears to be specifically recognized in a small pocket within the binding groove. Hence, the IF domain can preselect and prealign substrates with proline residues, which may explain how it enhances the catalytic efficiency and modulates the specificity of the FKBP domain in addition to its chaperone function. Based on pulldown results, we suggest that SlpA is likely to be involved in ribosome assembly.—Quistgaard, E. M., Nordlund, P., Löw, C. High-resolution insights into binding of unfolded polypeptides by the PPIase chaperone SlpA

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

Structural Basis for PTPA Interaction with the Invariant C-Terminal Tail of PP2A

Protein phosphatase 2A (PP2A) is a highly abundant heterotrimeric Ser/Thr phosphatase involved in the regulation of a variety of signaling pathways. The PP2A phosphatase activator (PTPA) is an ATP-dependent activation chaperone, which plays a key role in the biogenesis of active PP2A. The C-terminal tail of the catalytic subunit of PP2A is highly conserved and can undergo a number of posttranslational modifications that serve to regulate the function of PP2A. Here we have studied structurally the interaction of PTPA with the conserved C-terminal tail of the catalytic subunit carrying different posttranslational modifications. We have identified an additional interaction site for the invariant C-terminal tail of the catalytic subunit on PTPA, which can be modulated via posttranslational modifications. We show that phosphorylation of Tyr307PP2A-C or carboxymethylation of Leu309PP2A-C abrogates or diminishes binding of the C-terminal tail, whereas phosphorylation of Thr304PP2A-C is of no consequence. We suggest that the invariant C-terminal residues of the catalytic subunit can act as affinity enhancer for different PP2A interaction partners, including PTPA, and a different ‘code’ of posttranslational modifications can favour interactions to one subunit over others

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

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

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

Refinement statistics.

<p>Refinement statistics.</p