AlphaFold accurately predicts distinct conformations based on the oligomeric state of a de novo designed protein.

Using the molecular modeling program Rosetta, we designed a de novo protein, called SEWN0.1, which binds the heterotrimeric G protein Gαq. The design is helical, well-folded, and primarily monomeric in solution at a concentration of 10 µM. However, when we solved the crystal structure of SEWN0.1 at 1.9 Å, we observed a dimer in a conformation incompatible with binding Gαq . Unintentionally, we had designed a protein that adopts alternate conformations depending on its oligomeric state. Recently, there has been tremendous progress in the field of protein structure prediction as new methods in artificial intelligence have been used to predict structures with high accuracy. We were curious if the structure prediction method AlphaFold could predict the structure of SEWN0.1 and if the prediction depended on oligomeric state. When AlphaFold was used to predict the structure of monomeric SEWN0.1, it produced a model that resembles the Rosetta design model and is compatible with binding Gαq , but when used to predict the structure of a dimer, it predicted a conformation that closely resembles the SEWN0.1 crystal structure. AlphaFold's ability to predict multiple conformations for a single protein sequence should be useful for engineering protein switches.

Macromolecular modeling and design in Rosetta: recent methods and frameworks.

The Rosetta software for macromolecular modeling, docking and design is extensively used in laboratories worldwide. During two decades of development by a community of laboratories at more than 60 institutions, Rosetta has been continuously refactored and extended. Its advantages are its performance and interoperability between broad modeling capabilities. Here we review tools developed in the last 5 years, including over 80 methods. We discuss improvements to the score function, user interfaces and usability. Rosetta is available at http://www.rosettacommons.org.

Structure of an anti-PEG antibody reveals an open ring that captures highly flexible PEG polymers.

Polyethylene glycol (PEG) is a polymer routinely used to modify biologics and nanoparticles to prolong blood circulation and reduce immunogenicity of the underlying therapeutic. However, several PEGylated therapeutics induce the development of anti-PEG antibodies (APA), leading to reduced efficacy and increased adverse events. Given the highly flexible structure of PEG, how APA specifically bind PEG remains poorly understood. Here, we report a crystal structure illustrating the structural properties and conformation of the APA 6-3 Fab bound to the backbone of PEG. The structure reveals an open ring-like sub-structure in the Fab paratope, whereby PEG backbone is captured and then stabilized via Van der Waals interactions along the interior and exterior of the ring paratope surface. Our finding illustrates a strategy by which antibodies can bind highly flexible repeated structures that lack fixed conformations, such as polymers. This also substantially advances our understanding of the humoral immune response generated against PEG.

Engineering Polymer-Binding Bispecific Antibodies for Enhanced Pretargeted Delivery of Nanoparticles to Mucus-Covered Epithelium.

Mucus represents a major barrier to sustained and targeted drug delivery to mucosal epithelium. Ideal drug carriers should not only rapidly diffuse across mucus, but also bind the epithelium. Unfortunately, ligand-conjugated particles often exhibit poor penetration across mucus. In this work, we explored a two-step "pretargeting" approach through engineering a bispecific antibody that binds both cell-surface ICAM-1 and polyethylene glycol (PEG) on the surface of nanoparticles, thereby effectively decoupling cell targeting from particle design and formulation. When tested in a mucus-coated Caco-2 culture model that mimics the physiological process of mucus clearance, pretargeting increased the amount of PEGylated particles binding to cells by around 2-fold or more compared to either non-targeted or actively targeted PEGylated particles. Pretargeting also markedly enhanced particle retention in mouse intestinal tissues. Our work underscores pretargeting as a promising strategy to improve the delivery of therapeutics to mucosal surfaces.

UbSRD: The Ubiquitin Structural Relational Database.

The structurally defined ubiquitin-like homology fold (UBL) can engage in several unique protein-protein interactions and many of these complexes have been characterized with high-resolution techniques. Using Rosetta's structural classification tools, we have created the Ubiquitin Structural Relational Database (UbSRD), an SQL database of features for all 509 UBL-containing structures in the PDB, allowing users to browse these structures by protein-protein interaction and providing a platform for quantitative analysis of structural features. We used UbSRD to define the recognition features of ubiquitin (UBQ) and SUMO observed in the PDB and the orientation of the UBQ tail while interacting with certain types of proteins. While some of the interaction surfaces on UBQ and SUMO overlap, each molecule has distinct features that aid in molecular discrimination. Additionally, we find that the UBQ tail is malleable and can adopt a variety of conformations upon binding. UbSRD is accessible as an online resource at rosettadesign.med.unc.edu/ubsrd.

Design of Protein Multi-specificity Using an Independent Sequence Search Reduces the Barrier to Low Energy Sequences.

Computational protein design has found great success in engineering proteins for thermodynamic stability, binding specificity, or enzymatic activity in a 'single state' design (SSD) paradigm. Multi-specificity design (MSD), on the other hand, involves considering the stability of multiple protein states simultaneously. We have developed a novel MSD algorithm, which we refer to as REstrained CONvergence in multi-specificity design (RECON). The algorithm allows each state to adopt its own sequence throughout the design process rather than enforcing a single sequence on all states. Convergence to a single sequence is encouraged through an incrementally increasing convergence restraint for corresponding positions. Compared to MSD algorithms that enforce (constrain) an identical sequence on all states the energy landscape is simplified, which accelerates the search drastically. As a result, RECON can readily be used in simulations with a flexible protein backbone. We have benchmarked RECON on two design tasks. First, we designed antibodies derived from a common germline gene against their diverse targets to assess recovery of the germline, polyspecific sequence. Second, we design "promiscuous", polyspecific proteins against all binding partners and measure recovery of the native sequence. We show that RECON is able to efficiently recover native-like, biologically relevant sequences in this diverse set of protein complexes.

Data in support of UbSRD: The Ubiquitin Structural Relational Database.

This article provides information to support the database article titled "UbSRD: The Ubiquitin Structural Relational Database" (Harrison et al., 2015) [1] . The ubiquitin-like homology fold (UBL) represents a large family that encompasses both post-translational modifications, like ubiquitin (UBQ) and SUMO, and functional domains on many biologically important proteins like Parkin, UHRF1 (ubiquitin-like with PDB and RING finger domains-1), and Usp7 (ubiquitin-specific protease-7) (Zhang et al., 2015; Rothbart et al., 2013; Burroughs et al., 2012; Wauer et al., 2015) [2], [3], [4], [5]. The UBL domain can participate in several unique protein-protein interactions (PPI) since protein adducts can be attached to and removed from amino groups of lysine side chains and the N-terminus of proteins. Given the biological significance of UBL domains, many have been characterized with high-resolution techniques, and for UBQ and SUMO, many protein complexes have been characterized. We identified all the UBL domains in the PDB and created a relational database called UbSRD (Ubiquitin Structural Relational Database) by using structural analysis tools in the Rosetta (Leaver et al., 2013; O'Meara et al., 2015; Leaver-fay et al., 2011) [1], [6], [7], [8]. Querying UbSRD permitted us to report many quantitative properties of UBQ and SUMO recognition at different types interfaces (noncovalent: NC, conjugated: CJ, and deubiquitanse: DB). In this data article, we report the average number of non-UBL neighbors, secondary structure of interacting motifs, and the type of inter-molecular hydrogen bonds for each residue of UBQ and SUMO. Additionally, we used PROMALS3D to generate a multiple sequence alignment used to construct a phylogram for the entire set of UBLs (Pei and Grishin, 2014) [9]. The data described here will be generally useful to scientists studying the molecular basis for recognition of UBQ or SUMO.