Structural basis for substrate recognition, ligation and activation by a hyperactive Asn peptide ligase from Viola yedoensis

Peptide asparaginyl ligases (PALs) belong to a limited class of enzymes from cyclotide-producing plants, that perform site-specific ligation reactions after a target peptide Asx (Asn/Asp) binds to the ligase active site. How PALs specifically recognize their polypeptide substrates has remained elusive especially at the prime binding side of the enzyme. Here we captured VyPAL2, a catalytically efficient PAL from Viola yedoensis, in an activated state, with and without a bound substrate. The bound structure shows one ligase with the N-terminal polypeptide tail from another ligase molecule trapped at its active site, revealing how Asx inserts in the enzyme's S1 pocket and why a hydrophobic residue is required at the substrate P2' position. Beside illustrating the role played by P1 and P2' residues as primary anchors for the enzyme reaction, these results provide a mechanistic explanation for the role of the "Gatekeeper" residue at the surface of the S2 pocket, in shifting the non-prime portion of the substrate and, as a result, the activity towards either ligation or hydrolysis. These results detail the molecular events that occur during proenzyme maturation in the plant vacuolar compartment, suggest a mechanism for ligation, and will inform the design of peptide ligases with tailored specificities.

Analysis of the SNARE Stx8 recycling reveals that the retromer-sorting motif has undergone evolutionary divergence

Fsv1/Stx8 is a Schizosaccharomyces pombe protein similar to mammalian syntaxin 8. stx8Δ cells are sensitive to salts, and the prevacuolar endosome (PVE) is altered in stx8Δ cells. These defects depend on the SNARE domain, data that confirm the conserved function of syntaxin8 and Stx8 in vesicle fusion at the PVE. Stx8 localizes at the trans-Golgi network (TGN) and the prevacuolar endosome (PVE), and its recycling depends on the retromer component Vps35, and on the sorting nexins Vps5, Vps17, and Snx3. Several experimental approaches demonstrate that Stx8 is a cargo of the Snx3-retromer. Using extensive truncation and alanine scanning mutagenesis, we identified the Stx8 sorting signal. This signal is an IEMeaM sequence that is located in an unstructured protein region, must be distant from the transmembrane (TM) helix, and where the 133I, 134E, 135M, and 138M residues are all essential for recycling. This sorting motif is different from those described for most retromer cargoes, which include aromatic residues, and resembles the sorting motif of mammalian polycystin-2 (PC2). Comparison of Stx8 and PC2 motifs leads to an IEMxx(I/M) consensus. Computer-assisted screening for this and for a loose Ψ(E/D)ΨXXΨ motif (where Ψ is a hydrophobic residue with large aliphatic chain) shows that syntaxin 8 and PC2 homologues from other organisms bear variation of this motif. The phylogeny of the Stx8 sorting motifs from the Schizosaccharomyces species shows that their divergence is similar to that of the genus, showing that they have undergone evolutionary divergence. A preliminary analysis of the motifs in syntaxin 8 and PC2 sequences from various organisms suggests that they might have also undergone evolutionary divergence, what suggests that the presence of almost-identical motifs in Stx8 and PC2 might be a case of convergent evolution.

Alternative catalytic mechanisms driven by structural plasticity is an emerging theme in HAS-GTPases, Era and FeoB

GTP hydrolysis is the underlying basis for functioning of ‘biological switches’ or GTPases. Extensively studied GTPases, Ras and EF-Tu, use a conserved Gln/His that facilitates the activation of attacking water for nucleophilic attack. However, this is insufficient to explain catalysis in Hydrophobic Amino acid Substituted (HAS)-GTPases that naturally possess a hydrophobic residue in lieu of Gln/His. We had previously reported a bridging water-chain mediated catalytic mechanism for HAS-GTPase FeoB; which utilizes two distantly-located but conserved glutamates. Curiously, mutating these does not abolish GTP hydrolysis. Similarly, in this study we report our observations on another HAS-GTPase Era, wherein the mutants of catalytically important residues continue to hydrolyze GTP. We attempt to rationalize these inquisitive observations on GTP hydrolysis by FeoB and Era mutants. We propose a general theory that appears common to at least three classes of GTPases, where ‘alternative mechanisms’ emerge when the primary mechanism is disrupted. Based on the analysis of crystal structures of FeoB and Era mutants, bound to the transition state analogue GDP.AlFx, this work suggests that in the absence of catalytically important residues, the active site waters in both FeoB and Era undergo re-arrangements, which in turn helps in sustaining GTP hydrolysis. Similar employment of alternative mechanisms was also suggested for the catalytic mutants of hGBP1. Importantly, such alternatives underscore the robustness of GTP hydrolysis mechanisms in these systems, and raise important questions regarding the need for persistent GTP hydrolysis and the physiological relevance of structural plasticity seen here.

The structure of a potassium-selective ion channel reveals a hydrophobic gate regulating ion permeation

Protein dynamics are essential to function. One example of this is the various gating mechanisms within ion channels, which are transmembrane proteins that act as gateways into the cell. Typical ion channels switch between an open and closed state via a conformational transition which is often triggered by an external stimulus, such as ligand binding or pH and voltage differences. The atomic resolution structure of a potassium-selective ion channel named NaK2K has allowed us to observe that a hydrophobic residue at the bottom of the selectivity filter, Phe92, appears in dual conformations. One of the two conformations of Phe92 restricts the diameter of the exit pore around the selectivity filter, limiting ion flow through the channel, while the other conformation of Phe92 provides a larger-diameter exit pore from the selectivity filter. Thus, it can be concluded that Phe92 acts as a hydrophobic gate, regulating the flow of ions through the selectivity filter.

Structure of substrate-bound SMG1-8-9 kinase complex reveals molecular basis for phosphorylation specificity

PI3K-related kinases (PIKKs) are large Serine/Threonine (Ser/Thr)-protein kinases central to the regulation of many fundamental cellular processes. PIKK family member SMG1 orchestrates progression of an RNA quality control pathway, termed nonsense-mediated mRNA decay (NMD), by phosphorylating the NMD factor UPF1. Phosphorylation of UPF1 occurs in its unstructured N- and C-terminal regions at Serine/Threonine-Glutamine (SQ) motifs. How SMG1 and other PIKKs specifically recognize SQ motifs has remained unclear. Here, we present a cryo-electron microscopy (cryo-EM) reconstruction of a human SMG1-8-9 kinase complex bound to a UPF1 phosphorylation site at an overall resolution of 2.9 Å. This structure provides the first snapshot of a human PIKK with a substrate-bound active site. Together with biochemical assays, it rationalizes how SMG1 and perhaps other PIKKs specifically phosphorylate Ser/Thr-containing motifs with a glutamine residue at position +1 and a hydrophobic residue at position -1, thus elucidating the molecular basis for phosphorylation site recognition.

The Role of Hydrophobicity in the Stability and pH-Switchability of (RXDX)4 and Coumarin-RXDX Conjugate β-Sheets

<p>pH-switchable, self-assembling materials are of interest in biological imaging and sensing applications. Here we propose that combining the pH-switchability of RXDX (X=Ala, Val, Leu, Ile, Phe) peptides and the optical properties of coumarin creates an ideal candidate for these materials. This suggestion is tested with a thorough set of all-atom molecular dynamics simulations. We first investigate the dependence of pH-switchabiliy on the identity of the hydrophobic residue, X, in the bare (RXDX)₄ systems. Increasing the hydrophobicity stabilizes the fiber which, in turn, reduces the pH-switchabilty of the system. This behavior is found to be somewhat transferable to systems in which a single hydrophobic residue is replaced with a coumarin containing amino acid. In this case, conjugates with X=Ala are found to be unstable and both pHs while conjugates with X=Val, Leu, Ile and Phe are found to form stable β-sheets at least at neutral pH. The (RFDF)₄-coumarin conjugate is found to have the largest relative entropy value of 0.884 +/- 0.001 between neutral and acidic coumarin ordering distributions. Thus, we posit that coumarin-(RFDF)₄ containing peptide sequences are ideal candidates for pH-sensing bioelectronic materials.</p>

The Role of Hydrophobicity in the Stability and pH-Switchability of (RXDX)4 and Coumarin-RXDX Conjugate β-Sheets

<p>pH-switchable, self-assembling materials are of interest in biological imaging and sensing applications. Here we propose that combining the pH-switchability of RXDX (X=Ala, Val, Leu, Ile, Phe) peptides and the optical properties of coumarin creates an ideal candidate for these materials. This suggestion is tested with a thorough set of all-atom molecular dynamics simulations. We first investigate the dependence of pH-switchabiliy on the identity of the hydrophobic residue, X, in the bare (RXDX)₄ systems. Increasing the hydrophobicity stabilizes the fiber which, in turn, reduces the pH-switchabilty of the system. This behavior is found to be somewhat transferable to systems in which a single hydrophobic residue is replaced with a coumarin containing amino acid. In this case, conjugates with X=Ala are found to be unstable and both pHs while conjugates with X=Val, Leu, Ile and Phe are found to form stable β-sheets at least at neutral pH. The (RFDF)₄-coumarin conjugate is found to have the largest relative entropy value of 0.884 +/- 0.001 between neutral and acidic coumarin ordering distributions. Thus, we posit that coumarin-(RFDF)₄ containing peptide sequences are ideal candidates for pH-sensing bioelectronic materials.</p>

Molecular Basis for the Evolved Instability of a Human G-Protein Coupled Receptor

ABSTRACTMembrane proteins are prone to misfolding and degradation. This is particularly true for mammalian forms of the gonadotropin-releasing hormone receptor (GnRHR). Though they function at the plasma membrane, mammalian GnRHRs tend to accumulate within the secretory pathway. Their apparent instability is believed to have evolved in response to selection for attenuated GnRHR activity. Nevertheless, the structural basis of this adaptation remains unclear. We find that this adaptation coincides with a C-terminal truncation and an increase in the polarity of its transmembrane (TM) domains. This enhanced polarity compromises the translocon-mediated cotranslational folding of two TM domains. Moreover, replacing a conserved polar residue in TM6 with an ancestral hydrophobic residue partially restores GnRHR expression with minimal impact on function. An evolutionary analysis suggests variations in the polarity of this residue are associated with reproductive differences. Our findings suggest the marginal energetics of cotranslational folding can be exploited to tune membrane protein fitness.

Structural Conservation and Diversity of PilZ-Related Domains

ABSTRACT The widespread bacterial second messenger cyclic diguanylate (c-di-GMP) regulates a variety of processes, including protein secretion, motility, cell development, and biofilm formation. c-di-GMP-dependent responses are often mediated by its binding to the cytoplasmic receptors that contain the PilZ domain. Here, we present comparative structural and sequence analysis of various PilZ-related domains and describe three principal types of them: (i) the canonical PilZ domain, whose structure includes a six-stranded beta-barrel and a C-terminal alpha helix, (ii) an atypical PilZ domain that contains two extra alpha helices and forms stable tetramers, and (iii) divergent PilZ-related domains, which include the eponymous PilZ protein and PilZN (YcgR_N) and PilZNR (YcgR_2) domains. We refine the second c-di-GMP binding motif of PilZ as [D/N]hSXXG and show that the hydrophobic residue h of this motif interacts with a cluster of conserved hydrophobic residues, helping maintain the PilZ domain fold. We describe several novel PilZN-type domains that are fused to the canonical PilZ domains in specific taxa, such as spirochetes, actinobacteria, aquificae, cellulose-degrading clostridia, and deltaproteobacteria. We propose that the evolution of the three major groups of PilZ domains included (i) fusion of pilZ with other genes, which produced Alg44, cellulose synthase, and other multidomain proteins; (ii) insertion of an ∼200-bp fragment, which resulted in the formation of tetramer-forming PilZ proteins; and (iii) tandem duplication of pilZ genes, which led to the formation of PilZ dimers and YcgR-like proteins. IMPORTANCE c-di-GMP is a ubiquitous bacterial second messenger that regulates motility, biofilm formation, and virulence of many bacterial pathogens. The PilZ domain is a widespread c-di-GMP receptor that binds c-di-GMP through its RXXXR and [D/N]hSXXG motifs; some PilZ domains lack these motifs and are unable to bind c-di-GMP. We used structural and sequence analysis to assess the diversity of PilZ-related domains and define their common features. We show that the hydrophobic residue h in the second position of the second motif is highly conserved; it may serve as a readout for c-di-GMP binding. We describe three principal classes of PilZ-related domains, canonical, tetramer-forming, and divergent PilZ domains, and propose the evolutionary pathways that led to the emergence of these PilZ types.