Minimization of the E. coli ribosome, aided and optimized by community science.

The ribosome is a ribonucleoprotein complex found in all domains of life. Its role is to catalyze protein synthesis, the messenger RNA (mRNA)-templated formation of amide bonds between α-amino acid monomers. Amide bond formation occurs within a highly conserved region of the large ribosomal subunit known as the peptidyl transferase center (PTC). Here we describe the step-wise design and characterization of mini-PTC 1.1, a 284-nucleotide RNA that recapitulates many essential features of the Escherichia coli PTC. Mini-PTC 1.1 folds into a PTC-like structure under physiological conditions, even in the absence of r-proteins, and engages small molecule analogs of A- and P-site tRNAs. The sequence of mini-PTC 1.1 differs from the wild type E. coli ribosome at 12 nucleotides that were installed by a cohort of citizen scientists using the on-line video game Eterna. These base changes improve both the secondary structure and tertiary folding of mini-PTC 1.1 as well as its ability to bind small molecule substrate analogs. Here, the combined input from Eterna citizen-scientists and RNA structural analysis provides a robust workflow for the design of a minimal PTC that recapitulates many features of an intact ribosome.

Expanding the substrate scope of pyrrolysyl-transfer RNA synthetase enzymes to include non-α-amino acids in vitro and in vivo.

The absence of orthogonal aminoacyl-transfer RNA (tRNA) synthetases that accept non-L-α-amino acids is a primary bottleneck hindering the in vivo translation of sequence-defined hetero-oligomers and biomaterials. Here we report that pyrrolysyl-tRNA synthetase (PylRS) and certain PylRS variants accept α-hydroxy, α-thio and N-formyl-L-α-amino acids, as well as α-carboxy acid monomers that are precursors to polyketide natural products. These monomers are accommodated and accepted by the translation apparatus in vitro; those with reactive nucleophiles are incorporated into proteins in vivo. High-resolution structural analysis of the complex formed between one PylRS enzyme and a m-substituted 2-benzylmalonic acid derivative revealed an active site that discriminates prochiral carboxylates and accommodates the large size and distinct electrostatics of an α-carboxy substituent. This work emphasizes the potential of PylRS-derived enzymes for acylating tRNA with monomers whose α-substituent diverges substantially from the α-amine of proteinogenic amino acids. These enzymes or derivatives thereof could synergize with natural or evolved ribosomes and/or translation factors to generate diverse sequence-defined non-protein heteropolymers.

Chemsearch: collaborative compound libraries with structure-aware browsing.

Summary: Chemsearch is a cross-platform server application for developing and managing a chemical compound library and associated data files, with an interface for browsing and search that allows for easy navigation to a compound of interest, similar compounds or compounds that have desired structural properties. With provisions for access control and centralized document and data storage, Chemsearch supports collaboration by distributed teams. Availability and implementation: Chemsearch is a free and open-source Flask web application that can be linked to a Google Workspace account. Source code is available at https://github.com/gem-net/chemsearch (GPLv3 license). A Docker image allowing rapid deployment is available at https://hub.docker.com/r/cgemcci/chemsearch.

Modular HIV-1 Capsid Assemblies Reveal Diverse Host-Capsid Recognition Mechanisms.

The HIV-1 capsid is an ordered protein shell that houses the viral genome during early infection. Its expansive surface consists of an ordered and interfacing array of capsid protein hexamers and pentamers that are recognized by numerous cellular proteins. Many of these proteins recognize specific, assembled capsid interfaces not present in unassembled capsid subunits. We used protein-engineering tools to capture diverse capsid assembly intermediates. We built a repertoire of capsid assemblies (ranging from two to 42 capsid protein molecules) that recreate the various surfaces in infectious capsids. These assemblies reveal unique capsid-targeting mechanisms for each of the anti-HIV factors, TRIMCyp, MxB, and TRIM5α, linked to inhibition of virus uncoating and nuclear entry, as well as the HIV-1 cofactor FEZ1 that facilitates virus intracellular trafficking. This capsid assembly repertoire enables elucidation of capsid recognition modes by known capsid-interacting factors, identification of new capsid-interacting factors, and potentially, development of capsid-targeting therapeutics.

GEM-NET: Lessons in Multi-Institution Teamwork Using Collaboration Software.

The Center for Genetically Encoded Materials (C-GEM) is an NSF Phase I Center for Chemical Innovation that comprises six laboratories spread across three university campuses. Our success as a multi-institution research team demanded the development of a software infrastructure, GEM-NET, that allows all C-GEM members to work together seamlessly-as though everyone was in the same room. GEM-NET was designed to support both science and communication by integrating task management, scheduling, data sharing, and collaborative document and code editing with frictionless internal and public communication; it also maintains security over data and internal communications. In this Article, we document the design and implementation of GEM-NET: our objectives and motivating goals, how each component contributes to these goals, and the lessons learned throughout development. We also share open source code for several custom applications and document how GEM-NET can benefit users in multiple fields and teams that are both small and large. We anticipate that this knowledge will guide other multi-institution teams, regardless of discipline, to plan their software infrastructure and utilize it as swiftly and smoothly as possible.

MxB Restricts HIV-1 by Targeting the Tri-hexamer Interface of the Viral Capsid.

The human antiviral protein MxB is a restriction factor that fights HIV infection. Previous experiments have demonstrated that MxB targets the HIV capsid, a protein shell that protects the viral genome. To make the conical-shaped capsid, HIV CA proteins are organized into a lattice composed of hexamer and pentamer building blocks, providing many interfaces for host proteins to recognize. Through extensive biochemical and biophysical studies and molecular dynamics simulations, we show that MxB is targeting the HIV capsid by recognizing the region created at the intersection of three CA hexamers. We are further able to map this interaction to a few CA residues, located in a negatively charged well at the interface between the three CA hexamers. This work provides detailed residue-level mapping of the targeted capsid interface and how MxB interacts. This information could inspire the development of capsid-targeting therapies for HIV.

Cyanobacterial polyketide synthase docking domains: a tool for engineering natural product biosynthesis.

Modular type I polyketide synthases (PKSs) are versatile biosynthetic systems that initiate, successively elongate, and modify acyl chains. Intermediate transfer between modules is mediated via docking domains, which are attractive targets for PKS pathway engineering to produce natural product analogs. We identified a class 2 docking domain in cyanobacterial PKSs and determined crystal structures for two docking domain pairs, revealing a distinct class 2 docking strategy for promoting intermediate transfer. The selectivity of class 2 docking interactions, demonstrated in binding and biochemical assays, could be altered by mutagenesis. We determined the ideal fusion location for exchanging class 1 and class 2 docking domains and demonstrated effective polyketide chain transfer in heterologous modules. Thus, class 2 docking domains are tools for rational bioengineering of a broad range of PKSs containing either class 1 or 2 docking domains.