A Real-Time All-Atom Structural Search Engine for Proteins

<div><p>Protein designers use a wide variety of software tools for <i>de novo</i> design, yet their repertoire still lacks a fast and interactive all-atom search engine. To solve this, we have built the Suns program: a real-time, atomic search engine integrated into the PyMOL molecular visualization system. Users build atomic-level structural search queries within PyMOL and receive a stream of search results aligned to their query within a few seconds. This instant feedback cycle enables a new “designability”-inspired approach to protein design where the designer searches for and interactively incorporates native-like fragments from proven protein structures. We demonstrate the use of Suns to interactively build protein motifs, tertiary interactions, and to identify scaffolds compatible with hot-spot residues. The official web site and installer are located at <a href="http://www.degradolab.org/suns/" target="_blank">http://www.degradolab.org/suns/</a> and the source code is hosted at <a href="https://github.com/godotgildor/Suns" target="_blank">https://github.com/godotgildor/Suns</a> (PyMOL plugin, BSD license), <a href="https://github.com/Gabriel439/suns-cmd" target="_blank">https://github.com/Gabriel439/suns-cmd</a> (command line client, BSD license), and <a href="https://github.com/Gabriel439/suns-search" target="_blank">https://github.com/Gabriel439/suns-search</a> (search engine server, GPLv2 license).</p></div

Throughput benchmarks.

<p>We compare throughput of search queries for both Suns and Erebus, defined as query time divided by number of models in the data set. Suns throughput is measured against a locally hosted server and the Erebus throughput data is taken from <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003750#pcbi.1003750-Shirvanyants1" target="_blank">[12]</a>. Detailed query information, including the query size in atoms and the number of matches, is provided in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003750#pcbi.1003750.s003" target="_blank">Table S3</a> and the specific query PDB files are included in the benchmark suite of suns-cmd (<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003750#pcbi.1003750.s006" target="_blank">Software S2</a>).</p

Overview of Suns algorithm and architecture.

<p>(Inputs) The search index is built from two inputs: a set of words to recognize and a set of protein structures to search subdivided into pages. (Index) The two underlying data structures are a forward index that translates words to matching pages and a database of every page which translates matched words to atoms within each page. (Server) Each request to the server is broken into three steps: consult the forward index to find potentially matching pages, filter matching pages by RMSD to the query, and aligning successful matches to the query. (Queue) A message queue forwards requests from clients to servers, and forwards responses from servers to clients. (Clients) Suns provides two client interfaces: a PyMOL search plugin and the suns-cmd command line interface.</p

Building a tertiary interaction.

<p>(A) Three strands are seeded by searching on a valine, which reveals two nearby clusters of valine and tyrosine. (B) Strands are extended one residue in each direction by searching for pairs of residues (colored yellow) in the context of an insertion site, yielding clusters of potential inserts (colored green). (C) The final backbone fragment (green) is fed to MadCaT, which identifies multiple compatible scaffolds. One such scaffold (PDB ID = 1E54, colored light grey) possesses many exact residue/rotamer matches to the assembled fragment (blue highlights) and many close matches (yellow highlights) that differ by a related residue (threonine to serine or valine to isoleucine) or by varying the rotamer.</p

Incremental assembly of a motif.

<p>(A) An initial search for a guanidinium fragment reveals a cluster of nearby carboxylates. (B) Refining the search with one carboxylate from the results reveals a specific linker preference for both the aspartate and arginine involved in the salt bridge. (C) Adding the most common linker for arginine and repeating the search reveals that this salt bridge is part of an N-terminal capping motif. Search queries are represented as thick sticks and search results are shown as thin lines. Grey dashed lines highlight search queries and black dashed lines highlight clusters in the search results, which are filtered to show the specific residue fragments of interest and neighboring water molecules within 3.0 Å as red spheres. Search parameters and fragments listed in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003750#pcbi.1003750.s002" target="_blank">Table S2</a>.</p

Searching for calcium binding sites.

<p>(A) Two side chains of the EF-hand of calmodulin suffice to find matching motifs. The search query (black dashes) consists exclusively of two aspartate side chains (D20 and D24) and does not include the calcium ligand. (B) Searching for these two side chains at 0.7 Å resolution returns seven results, all of which are EF-hand motifs. Six of these motifs coordinate a matching calcium ion (green sphere), and the seventh motif coordinates a sodium ion (purple sphere).</p

Water Distribution, Dynamics, and Interactions with Alzheimer’s β‑Amyloid Fibrils Investigated by Solid-State NMR

Water is essential for protein folding and assembly of amyloid fibrils. Internal water cavities have been proposed for several amyloid fibrils, but no direct structural and dynamical data have been reported on the water dynamics and site-specific interactions of water with the fibrils. Here we use solid-state NMR spectroscopy to investigate the water interactions of several Aβ40 fibrils. ¹H spectral lineshapes, T₂ relaxation times, and two-dimensional (2D) ¹H–¹³C correlation spectra show that there are five distinct water pools: three are peptide-bound water, while two are highly dynamic water that can be assigned to interfibrillar water and bulk-like matrix water. All these water pools are associated with the fibrils on the nanometer scale. Water-transferred 2D correlation spectra allow us to map out residue-specific hydration and give evidence for the presence of a water pore in the center of the three-fold symmetric wild-type Aβ40 fibril. In comparison, the loop residues and the intramolecular strand–strand interface have low hydration, excluding the presence of significant water cavities in these regions. The Osaka Aβ40 mutant shows lower hydration and more immobilized water than wild-type Aβ40, indicating the influence of peptide structure on the dynamics and distribution of hydration water. Finally, the highly mobile interfibrillar and matrix water exchange with each other on the time scale of seconds, suggesting that fibril bundling separates these two water pools, and water molecules must diffuse along the fibril axis before exchanging between these two environments. These results provide insights and experimental constraints on the spatial distribution and dynamics of water pools in these amyloid fibrils

The expression profile of M2 protein and AcGFP-M2 fusion protein.

<p>(<b>A</b>) Expression of AcGFP-M2 fusion protein in the presence or absence of amantadine. Experiment procedures are the same as described in a. Positions of M2 protein and AcGFP-M2 fusion protein are indicated by arrowheads. (B) Expression of M2 protein in the presence or absence of amantadine. Cells harboring pColdII(sp-4) <i>m2</i> were cultured as described previously until O.D.₆₀₀ reached 0.5–0.6 and incubated at 15°C for 45 to 60 mins. 1 mM IPTG was added to at 5-hr and the culture was divided into two, one of which contains 50 μM amantadine. Cells from 1.5 ml culture were collected at 0 hr, 1 hr, 3 hr, 5 hr, 7 hr, and 19 hr after induction and subjected to SDS-PAGE assay.</p

Discovery of Highly Potent Inhibitors Targeting the Predominant Drug-Resistant S31N Mutant of the Influenza A Virus M2 Proton Channel

With the emergence of highly pathogenic avian influenza (HPAI) H7N9 and H5N1 strains, there is a pressing need to develop direct-acting antivirals (DAAs) to combat such deadly viruses. The M2-S31N proton channel of the influenza A virus (A/M2) is one of the validated and most conserved proteins encoded by the current circulating influenza A viruses; thus, it represents a high-profile drug target for therapeutic intervention. We recently discovered a series of S31N inhibitors with the general structure of adamantyl-1-NH₂⁺CH₂–aryl, but they generally had poor physical properties and some showed toxicity in vitro. In this study, we sought to optimize both the adamantyl as well as the aryl/heteroaryl group. Several compounds from this study exhibited submicromolar EC₅₀ values against S31N-containing A/WSN/33 influenza viruses in antiviral plaque reduction assays with a selectivity index greater than 100, indicating that these compounds are promising candidates for in-depth preclinical pharmacology

Inhibition of M2 channel activity in Oocyte assay.

<p>The inhibition efficiency of each compound was examined at the concentration of 100 μM. The values in the table represent how much the M2 channel activity is inhibited. WT, wild type M2 channel. V27A, M2 channel with V27 A mutation. The Oocyte assay was conducted as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054070#pone.0054070-Balannik2" target="_blank">[12]</a>.</p