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Biomolecular structure drives function, and computational capabilities have progressed such that the prediction and computational design of biomolecular structures is increasingly feasible. Because computational biophysics attracts students from many different backgrounds and with different levels of resources, teaching the subject can be challenging. One strategy to teach diverse learners is with interactive multimedia material that promotes self-paced, active learning. We have created a hands-on education strategy with a set of fifteen modules that teach topics in biomolecular structure and design, from fundamentals of conformational sampling and energy evaluation to applications like protein docking, antibody design, and RNA structure prediction. Our modules are based on PyRosetta, a Python library that encapsulates all computational modules and methods in the Rosetta software package. The workshop-style modules are implemented as Jupyter Notebooks that can be executed in the Google Colaboratory, allowing learners access with just a web browser. The digital format of Jupyter Notebooks allows us to embed images, molecular visualization movies, and interactive coding exercises. This multimodal approach may better reach students from different disciplines and experience levels as well as attract more researchers from smaller labs and cognate backgrounds to leverage PyRosetta in their science and engineering research. All materials are freely available at https://github.com/RosettaCommons/PyRosetta.notebooks.

Optogenetic techniques use light-responsive proteins to study dynamic processes in living cells and organisms. These techniques typically rely on repurposed naturally occurring light-sensitive proteins to control sub-cellular localization and activity. We previously engineered two optogenetic systems, the Light Activated Nuclear Shuttle (LANS) and the Light-Inducible Nuclear eXporter (LINX), by embedding nuclear import or export sequence motifs into the C-terminal helix of the light-responsive LOV2 domain of Avena sativa phototropin 1, thus enabling light-dependent trafficking of a target protein into and out of the nucleus. While LANS and LINX are effective tools, we posited that mutations within the LOV2 hinge-loop, which connects the core PAS domain and the C-terminal helix, would further improve the functionality of these switches. Here, we identify hinge-loop mutations that favourably shift the dynamic range (the ratio of the on- to off-target subcellular accumulation) of the LANS and LINX photoswitches. We demonstrate the utility of these new optogenetic tools to control gene transcription and epigenetic modifications, thereby expanding the optogenetic ‘tool kit’ for the research community.

UHRF1 is a histone- and DNA-binding E3 ubiquitin ligase that functions with DNMT1 to maintain mammalian DNA methylation. UHRF1 facilitates DNMT1 recruitment to replicating chromatin through a coordinated mechanism involving histone and DNA recognition and histone ubiquitination. UHRF2 shares structural homology with UHRF1, but surprisingly lacks functional redundancy to facilitate DNA methylation maintenance. Molecular mechanisms uncoupling UHRF2 from DNA methylation maintenance are poorly defined. Through comprehensive and comparative biochemical analysis of recombinant human UHRF1 and UHRF2 reader and writer activities, we reveal conserved modes of histone PTM recognition but divergent DNA binding properties. While UHRF1 and UHRF2 diverge in their affinities toward hemi-methylated DNA, we surprisingly show that both hemi-methylated and hemi-hydroxymethylated DNA oligonucleotides stimulate UHRF2 ubiquitin ligase activity toward histone H3 peptide substrates. This is the first example of an E3 ligase allosterically regulated by DNA hydroxymethylation. However, UHRF2 is not a productive histone E3 ligase toward purified mononucleosomes, suggesting UHRF2 has an intra-domain architecture distinct from UHRF1 that is conformationally constrained when bound to chromatin. Collectively, our studies reveal that uncoupling of UHRF2 from the DNA methylation maintenance program is linked to differences in the molecular readout of chromatin signatures that connect UHRF1 to ubiquitination of histone H3.

During signal transduction, the G protein, Gαq, binds and activates phospholipase C-β isozymes. Several diseases have been shown to manifest upon constitutively activating mutation of Gαq, such as uveal melanoma. Therefore, methods are needed to directly inhibit Gαq. Previously, we demonstrated that a peptide derived from a helix-turn-helix (HTH) region of PLC-β3 (residues 852-878) binds Gαq with low micromolar affinity and inhibits Gαq by competing with full-length PLC-β isozymes for binding. Since the HTH peptide is unstructured in the absence of Gαq, we hypothesized that embedding the HTH in a folded protein might stabilize the binding-competent conformation and further improve the potency of inhibition. Using the molecular modeling software Rosetta, we searched the Protein Data Bank for proteins with similar HTH structures near their surface. The candidate proteins were computationally docked against Gαq and their surfaces were redesigned to stabilize this interaction. We then used yeast surface display to affinity mature the designs. The most potent design bound Gαq/i with high affinity in vitro (KD = 18 nM) and inhibited activation of PLC-β isozymes in HEK293 cells. We anticipate that our genetically encoded inhibitor will help interrogate the role of Gαq in healthy and disease model systems. Our work demonstrates that grafting interaction motifs into folded proteins is a powerful approach for generating inhibitors of protein-protein interactions.

SxIP-iLID is a novel optogenetic tool designed to assess the temporal role of proteins on microtubule dynamics. The authors establish that optogenetic cross-linking of microtubule and actin networks decreases MT growth velocities and increases the cell area void of microtubules., We developed a novel optogenetic tool, SxIP–improved light-inducible dimer (iLID), to facilitate the reversible recruitment of factors to microtubule (MT) plus ends in an end-binding protein–dependent manner using blue light. We show that SxIP-iLID can track MT plus ends and recruit tgRFP-SspB upon blue light activation. We used this system to investigate the effects of cross-linking MT plus ends and F-actin in Drosophila melanogaster S2 cells to gain insight into spectraplakin function and mechanism. We show that SxIP-iLID can be used to temporally recruit an F-actin binding domain to MT plus ends and cross-link the MT and F-actin networks. Cross-linking decreases MT growth velocities and generates a peripheral MT exclusion zone. SxIP-iLID facilitates the general recruitment of specific factors to MT plus ends with temporal control enabling researchers to systematically regulate MT plus end dynamics and probe MT plus end function in many biological processes., Graphical Abstract

Sactipeptides are ribosomally-synthesized peptides that contain a characteristic thioether bridge (sactionine bond) that is installed posttranslationally and is absolutely required for their antibiotic activity. Sactipeptide biosynthesis requires a unique family of radical SAM enzymes, which contain multiple [4Fe-4S] clusters, to form the requisite thioether bridge between a cysteine and the α-carbon of an opposing amino acid through radical-based chemistry. Here we present the structure of the sactionine bond-forming enzyme CteB, from Clostridium thermocellum ATCC 27405, with both SAM and an N-terminal fragment of its peptidyl-substrate at 2.04 Å resolution. CteB has the (β/α)(6)-TIM barrel fold that is characteristic of radical SAM enzymes, as well as a C-terminal SPASM domain that contains two auxiliary [4Fe-4S] clusters. Importantly, one [4Fe-4S] cluster in the SPASM domain exhibits an open coordination site in absence of peptide substrate, which is coordinated by a peptidyl-cysteine residue in the bound state. The crystal structure of CteB also reveals an accessory N-terminal domain that has high structural similarity to a recently discovered motif present in several enzymes that act on ribosomally-synthesized and post-translationally modified peptides (RiPPs), known as a RiPP precursor peptide recognition element (RRE). This crystal structure is the first of a sactionine bond forming enzyme and sheds light on structures and mechanisms of other members of this class such as AlbA or ThnB.

Light-sensitive proteins can be used to perturb signaling networks in living cells and animals with high spatiotemporal resolution. We recently engineered a protein heterodimer that dissociates when irradiated with blue light and demonstrated that by fusing each half of the dimer to termini of a protein that it is possible to selectively block binding surfaces on the protein when in the dark. Upon activation with light, the dimer dissociates and exposes the binding surface, allowing the protein to bind its partner. Critical to the success of this system, called Z-lock, is that the linkers connecting the dimer components to the termini are engineered so that the dimer forms over the appropriate binding surface. Here, we develop and test a protocol in the Rosetta molecular modeling program for designing linkers for Z-lock. We show that the protocol can predict the most effective linker sets for three different light-sensitive switches, including a newly designed switch that binds the Rho-family GTPase Cdc42 upon stimulation with blue light. This protocol represents a generalized computational approach to placing a wide variety of proteins under optogenetic control with Z-lock.

We used the molecular modeling program Rosetta to identify clusters of amino acid substitutions in antibody fragments (scFvs and scAbs) that improve global protein stability and resistance to thermal deactivation. Using this methodology, we increased the melting temperature (T(m)) and resistance to heat treatment of an antibody fragment that binds to the Clostridium botulinum hemagglutinin protein (anti-HA33). Two designed antibody fragment variants with two amino acid replacement clusters, designed to stabilize local regions, were shown to have both higher T(m) compared to the parental scFv and importantly, to retain full antigen binding activity after 2 hours of incubation at 70 °C. The crystal structure of one thermostabilized scFv variants was solved at 1.6 Å and shown to be in close agreement with the RosettaAntibody model prediction.

Proteins perform an amazingly diverse set of functions in all aspects of life. Critical to the function of many proteins are the highly specific three-dimensional structures they adopt. For this reason, there is strong interest in learning how to rationally design proteins that adopt user-defined structures. Over the last 25 years, there has been significant progress in the field of computational protein design as rotamer-based sequence optimization protocols have enabled accurate design of protein tertiary and quaternary structure. In this award article, I will summarize how the molecular modeling program Rosetta is used to design new protein structures and describe how we have taken advantage of this capability to create proteins that have important applications in research and medicine. I will highlight three protein design stories: the use of protein interface design to create therapeutic bispecific antibodies, the engineering of light-inducible proteins that can be used to recruit proteins to specific locations in the cell, and the de novo design of new protein structures from pieces of naturally occurring proteins.

The prediction of protein three-dimensional structure from amino acid sequence has been a grand challenge problem in computational biophysics for decades, owing to its intrinsic scientific interest and also to the many potential applications for robust protein structure prediction algorithms, from genome interpretation to protein function prediction. More recently, the inverse problem - designing an amino acid sequence that will fold into a specified three-dimensional structure - has attracted growing attention as a potential route to the rational engineering of proteins with functions useful in biotechnology and medicine. Methods for the prediction and design of protein structures have advanced dramatically in the past decade. Increases in computing power and the rapid growth in protein sequence and structure databases have fuelled the development of new data-intensive and computationally demanding approaches for structure prediction. New algorithms for designing protein folds and protein-protein interfaces have been used to engineer novel high-order assemblies and to design from scratch fluorescent proteins with novel or enhanced properties, as well as signalling proteins with therapeutic potential. In this Review, we describe current approaches for protein structure prediction and design and highlight a selection of the successful applications they have enabled.

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 ., This Perspective reviews tools developed over the past five years in the macromolecular modeling, docking and design software Rosetta.

Cells have evolved a variety of mechanisms to regulate the enormous complexity of processes taking place inside them. One mechanism consists in tightly controlling the localization of macromolecules, keeping them away from their place of action until needed. Since a large fraction of the cellular response to external stimuli is mediated by gene expression, it is not surprising that transcriptional regulators are often subject to stimulus-induced nuclear import or export. Here we review recent methods in chemical biology and optogenetics for controlling the nuclear localization of proteins of interest inside living cells. These methods allow researchers to regulate protein activity with exquisite spatiotemporal control, and open up new possibilities for studying the roles of proteins in a broad array of cellular processes and biological functions.

Protein ubiquitination involves E1, E2, and E3 trienzyme cascades. E2 and RING E3 enzymes often collaborate to first prime a substrate with a single ubiquitin (UB) and then achieve different forms of polyubiquitination: multiubiquitination of several sites and elongation of linkage-specific UB chains. Here, cryo-EM and biochemistry show that the human E3 anaphase-promoting complex/cyclosome (APC/C) and its two partner E2s, UBE2C (aka UBCH10) and UBE2S, adopt specialized catalytic architectures for these two distinct forms of polyubiquitination. The APC/C RING constrains UBE2C proximal to a substrate and simultaneously binds a substrate-linked UB to drive processive multiubiquitination. Alternatively, during UB chain elongation, the RING does not bind UBE2S but rather lures an evolving substrate-linked UB to UBE2S positioned through a cullin interaction to generate a Lys11-linked chain. Our findings define mechanisms of APC/C regulation, and establish principles by which specialized E3-E2-substrate-UB architectures control different forms of polyubiquitination.

Building new proteins from the old Proteins are the workhorses of biology. Designing new, stable proteins with functions desirable in biotechnology or biomedicine remains challenging. Jacobs et al. developed a computational method called SEWING that designs proteins using pieces of existing structures (see the Perspective by Netzer and Fleishman). The new proteins can contain structural features such as pockets or grooves that are required for function. The solved structures of two designed proteins agreed well with the design models. The method allows rapid design of a diverse set of structures that will facilitate functional design. Science , this issue p. 687 ; see also p. 657

Evolution trains a from-scratch catalyst Metal-bound peptides can catalyze simple reactions such as ester hydrolysis and may have been the starting point for the evolution of modern enzymes. Studer et al. selected progressively more-proficient variants of a small protein derived from a computationally designed zinc-binding peptide. The resulting enzyme could perform the trained reaction at rates typical for naturally evolved enzymes and serendipitously developed a strong preference for a single enantiomer of the substrate. A structure of the final catalyst highlights how small, progressive changes can remodel both catalytic residues and protein architecture in unpredictable ways. Science , this issue p. 1285

Protein binding specificities can be manipulated by redesigning contacts that already exist at an interface or by expanding the interface to allow interactions with residues adjacent to the original binding site. Previously, we developed a strategy, called AnchorDesign, for expanding interfaces around linear binding epitopes. The epitope is embedded in a loop of a scaffold protein, in our case a monobody, and then surrounding residues on the monobody are optimized for binding using directed evolution or computational design. Using this strategy, we have increased binding affinities by over 100-fold, but we have not tested whether it can be used to control protein binding specificities. Here, we test whether AnchorDesign can be used to engineer a monobody that binds specifically to the Mitogen-activated protein kinase (MAPK) p38α, but not to the related MAPKs ERK2 and JNK. To anchor the binding interaction, we used a small (D) docking motif from the Mitogen-activated protein kinase kinase (MAP2K) MKK6 that interacts with similar affinity to p38α and ERK2. Our hypothesis was that by embedding the motif in a larger protein that we could expand the interface and create contacts with residues that are not conserved between p38α and ERK2. Molecular modeling was used to inform insertion of the D motif into the monobody and a combination of phage and yeast display were used to optimize the interface. Binding experiments demonstrate that the engineered monobody binds to the target surface on p38α and does not exhibit detectable binding to ERK2 or JNK.

The spread of dengue (DENV) and Zika virus (ZIKV) is a major public health concern. The primary target of antibodies that neutralize DENV and ZIKV is the envelope (E) glycoprotein, and there is interest in using soluble recombinant E (sRecE) proteins as subunit vaccines. However, the most potent neutralizing antibodies against DENV and ZIKV recognize epitopes on the virion surface that span two or more E proteins. Therefore, to create effective DENV and ZIKV vaccines, presentation of these quaternary epitopes may be necessary. The sRecE proteins from DENV and ZIKV crystallize as native-like dimers, but studies in solution suggest that these dimers are marginally stable. To better understand the challenges associated with creating stable sRecE dimers, we characterized the thermostability of sRecE proteins from ZIKV and three DENV serotypes, DENV2-4. All four proteins irreversibly unfolded at moderate temperatures (46-53 °C). At 23 °C and low micromolar concentrations, DENV2 and ZIKV were primarily dimeric, and DENV3-4 were primarily monomeric, whereas at 37 °C, all four proteins were predominantly monomeric. We further show that the dissociation constant for DENV2 dimerization is very temperature-sensitive, ranging from

We have developed a set of protocols in the molecular modeling program Rosetta for performing requirement-driven protein design. First, the user specifies a set of structural features that need to be present in the designed protein. These requirements can be general (e.g., "create a protein with five helices"), or they can be very specific and require the correct placement of a set of amino acids to bind a ligand. Next, a large set of protein models are generated that satisfy the design requirements. The models are built using a method that we recently introduced into Rosetta, called SEWING, that rapidly assembles novel protein backbones by combining pieces of naturally occurring proteins. In the last step of the process, rotamer-based sequence optimization and backbone refinement are performed with Rosetta, and a variety of quality metrics are used to pick sequences for experimental characterization. Here we describe the input files and user options needed to run SEWING and perform requirement-driven design and provide detailed instructions for two specific applications of the process: the design of new structural elements at a protein-protein interface and the design of ligand binding sites.

Hydrogen bond networks play a critical role in determining the stability and specificity of biomolecular complexes, and the ability to design such networks is important for engineering novel structures, interactions, and enzymes. One key feature of hydrogen bond networks that makes them difficult to rationally engineer is that they are highly cooperative and are not energetically favorable until the hydrogen bonding potential has been satisfied for all buried polar groups in the network. Existing computational methods for protein design are ill-equipped for creating these highly cooperative networks because they rely on energy functions and sampling strategies that are focused on pairwise interactions. To enable the design of complex hydrogen bond networks, we have developed a new sampling protocol in the molecular modeling program Rosetta that explicitly searches for sets of amino acid mutations that can form self-contained hydrogen bond networks. For a given set of designable residues, the protocol often identifies many alternative sets of mutations/networks, and we show that it can readily be applied to large sets of residues at protein-protein interfaces or in the interior of proteins. The protocol builds on a recently developed method in Rosetta for designing hydrogen bond networks that has been experimentally validated for small symmetric systems but was not extensible to many larger protein structures and complexes. The sampling protocol we describe here not only recapitulates previously validated designs with performance improvements but also yields viable hydrogen bond networks for cases where the previous method fails, such as the design of large, asymmetric interfaces relevant to engineering protein-based therapeutics.

β‐sheets often have one face packed against the core of the protein and the other facing solvent. Mutational studies have indicated that the solvent‐facing residues can contribute significantly to protein stability, and that the preferred amino acid at each sequence position is dependent on the precise structure of the protein backbone and the identity of the neighboring amino acids. This suggests that the most advantageous methods for designing β‐sheet surfaces will be approaches that take into account the multiple energetic factors at play including side chain rotamer preferences, van der Waals forces, electrostatics, and desolvation effects. Here, we show that the protein design software Rosetta, which models these energetic factors, can be used to dramatically increase protein stability by optimizing interactions on the surfaces of small β‐sheet proteins. Two design variants of the β‐sandwich protein from tenascin were made with 7 and 14 mutations respectively on its β‐sheet surfaces. These changes raised the thermal midpoint for unfolding from 45°C to 64°C and 74°C. Additionally, we tested an empirical approach based on increasing the number of potential salt bridges on the surfaces of the β‐sheets. This was not a robust strategy for increasing stability, as three of the four variants tested were unfolded.

Light-inducible dimers are powerful tools for cellular optogenetics, as they can be used to control the localization and activity of proteins with high spatial and temporal resolution. Despite the generality of the approach, application of light-inducible dimers is not always straightforward, as it is frequently necessary to test alternative dimer systems and fusion strategies before the desired biological activity is achieved. This process is further hindered by an incomplete understanding of the biophysical/biochemical mechanisms by which available dimers behave and how this correlates to in vivo function. To better inform the engineering process, we examined the biophysical and biochemical properties of three blue-light-inducible dimer variants (cryptochrome2 (CRY2)/CIB1, iLID/SspB, and LOVpep/ePDZb) and correlated these characteristics to in vivo colocalization and functional assays. We find that the switches vary dramatically in their dark and lit state binding affinities and that these affinities correlate with activity changes in a variety of in vivo assays, including transcription control, intracellular localization studies, and control of GTPase signaling. Additionally, for CRY2, we observe that light-induced changes in homo-oligomerization can have significant effects on activity that are sensitive to alternative fusion strategies.

Summary Dendritic spines are the major loci of synaptic plasticity and are considered as possible structural correlates of memory. Nonetheless, systematic manipulation of specific subsets of spines in the cortex has been unattainable, and thus, the link between spines and memory has been correlational. We developed a novel synaptic optoprobe, AS-PaRac1 (activated synapse targeting photoactivatable Rac1), which can label recently potentiated spines specifically, and induce the selective shrinkage of AS-PaRac1-containing spines. In vivo imaging of AS-PaRac1 revealed that a motor learning induced substantial synaptic remodelling in a small subset of neurons. The acquired motor learning was disrupted by the optical shrinkage of the potentiated spines, whereas it was not affected by the identical manipulation of spines evoked by a distinct motor task in the same cortical region. Taken together, our results demonstrate that a newly acquired motor skill depends on the formation of a task-specific dense synaptic ensemble.

We engineered a photoactivatable system for rapidly and reversibly exporting proteins from the nucleus by embedding a nuclear export signal in the LOV2 domain from phototropin 1. Fusing the chromatin modifier Bre1 to the photoswitch, we achieved light-dependent control of histone H2B monoubiquitylation in yeast, revealing fast turnover of the ubiquitin mark. Moreover, this inducible system allowed us to dynamically monitor the status of epigenetic modifications dependent on H2B ubiquitylation.

Lipoprotein lipase (LPL) is a dimeric enzyme that is responsible for clearing triglyceride-rich lipoproteins from the blood. Although LPL plays a key role in cardiovascular health, an experimentally derived three-dimensional structure has not been determined. Such a structure would aid in understanding mutations in LPL that cause familial LPL deficiency in patients and help in the development of therapeutic strategies to target LPL. A major obstacle to structural studies of LPL is that LPL is an unstable protein that is difficult to produce in the quantities needed for nuclear magnetic resonance or crystallography. We present updated LPL structural models generated by combining disulfide mapping, computational modeling, and data derived from single-molecule Förster resonance energy transfer (smFRET). We pioneer the technique of smFRET for use with LPL by developing conditions for imaging active LPL and identifying positions in LPL for the attachment of fluorophores. Using this approach, we measure LPL-LPL intermolecular interactions to generate experimental constraints that inform new computational models of the LPL dimer structure. These models suggest that LPL may dimerize using an interface that is different from the dimerization interface suggested by crystal packing contacts seen in structures of pancreatic lipase.

Cellular signal transduction is often regulated at multiple steps in order to achieve more complex logic or precise control of a pathway. For instance, some signaling mechanisms couple allosteric activation with localization to achieve high signal to noise. Here, we create a system for light activated nuclear import that incorporates two levels of control. It consists of a nuclear import photoswitch, Light Activated Nuclear Shuttle (LANS), and a protein engineered to preferentially interact with LANS in the dark, Zdk2. First, Zdk2 is tethered to a location in the cytoplasm, which sequesters LANS in the dark. Second, LANS incorporates a nuclear localization signal (NLS) that is sterically blocked from binding to the nuclear import machinery in the dark. When activated with light, LANS both dissociates from its tethered location and exposes its NLS, which leads to nuclear accumulation. We demonstrate that this coupled system improves the dynamic range of LANS in mammalian cells, yeast, and C. elegans and provides tighter control of transcription factors that have been fused to LANS.

The use of bispecific antibodies (BsAbs) to treat human diseases is on the rise. Increasingly complex and powerful therapeutic mechanisms made possible by BsAbs are spurring innovation of novel BsAb formats and methods for their production. The long-lived in vivo pharmacokinetics, optimal biophysical properties and potential effector functions of natural IgG monoclonal (and monospecific) antibodies has resulted in a push to generate fully IgG BsAb formats with the same quaternary structure as monoclonal IgGs. The production of fully IgG BsAbs is challenging because of the highly heterogeneous pairing of heavy chains (HCs) and light chains (LCs) when produced in mammalian cells with two IgG HCs and two LCs. A solution to the HC heterodimerization aspect of IgG BsAb production was first discovered two decades ago; however, addressing the LC mispairing issue has remained intractable until recently. Here, we use computational and rational engineering to develop novel designs to the HC/LC pairing issue, and particularly for κ LCs. Crystal structures of these designs highlight the interactions that provide HC/LC specificity. We produce and characterize multiple fully IgG BsAbs using these novel designs. We demonstrate the importance of specificity engineering in both the variable and constant domains to achieve robust HC/LC specificity within all the BsAbs. These solutions facilitate the production of fully IgG BsAbs for clinical use.

The discovery of light-inducible protein-protein interactions has allowed for the spatial and temporal control of a variety of biological processes. To be effective, a photodimerizer should have several characteristics: it should show a large change in binding affinity upon light stimulation, it should not cross-react with other molecules in the cell, and it should be easily used in a variety of organisms to recruit proteins of interest to each other. To create a switch that meets these criteria we have embedded the bacterial SsrA peptide in the C-terminal helix of a naturally occurring photoswitch, the light-oxygen-voltage 2 (LOV2) domain from Avena sativa. In the dark the SsrA peptide is sterically blocked from binding its natural binding partner, SspB. When activated with blue light, the C-terminal helix of the LOV2 domain undocks from the protein, allowing the SsrA peptide to bind SspB. Without optimization, the switch exhibited a twofold change in binding affinity for SspB with light stimulation. Here, we describe the use of computational protein design, phage display, and high-throughput binding assays to create an improved light inducible dimer (iLID) that changes its affinity for SspB by over 50-fold with light stimulation. A crystal structure of iLID shows a critical interaction between the surface of the LOV2 domain and a phenylalanine engineered to more tightly pin the SsrA peptide against the LOV2 domain in the dark. We demonstrate the functional utility of the switch through light-mediated subcellular localization in mammalian cell culture and reversible control of small GTPase signaling.

The de novo design of proteins is a rigorous test of our understanding of the key determinants of protein structure. The helix bundle is an interesting de novo design model system due to the diverse topologies that can be generated from a few simple α-helices. Previously, noncomputational studies demonstrated that connecting amphipathic helices together with short loops can sometimes generate helix bundle proteins, regardless of the bundle's exact sequence. However, using such methods, the precise positions of helices and side chains cannot be predetermined. Since protein function depends on exact positioning of residues, we examined if sequence design tools in the program Rosetta could be used to design a four-helix bundle with a predetermined structure. Helix position was specified using a folding procedure that constrained the design model to a defined topology, and iterative rounds of rotamer-based sequence design and backbone refinement were used to identify a low energy sequence for characterization. The designed protein, DND_4HB, unfolds cooperatively (Tm >90°C) and a NMR solution structure shows that it adopts the target helical bundle topology. Helices 2, 3, and 4 agree very closely with the design model (backbone RMSD = 1.11 A) and >90% of the core side chain χ1 and χ2 angles are correctly predicted. Helix 1 lies in the target groove against the other helices, but is displaced 3 A along the bundle axis. This result highlights the potential of computational design to create bundles with atomic-level precision, but also points at remaining challenges for achieving specific positioning between amphipathic helices.

The computer-based design of PPIs (protein–protein interactions) is a challenging problem because large desolvation and entropic penalties must be overcome by the creation of favourable hydrophobic and polar contacts at the target interface. Indeed, many computationally designed interactions fail to form when tested in the laboratory. In the present article, we highlight strategies our laboratory has been pursuing to make interface design more tractable. Our general approach has been to make use of structural motifs found in native proteins that are predisposed to interact with a particular binding geometry, and then further bolster these anchor points with favourable hydrophobic contacts. We describe the use of three different anchor points, i.e. β-strand pairing, metal binding and the docking of α-helix into a groove, to successfully design new interfaces. In several cases, high-resolution crystal structures show that the design models closely match the experimental structure. In addition, we have tested the use of buried hydrogen-bond networks as a source of affinity and specificity at interfaces. In these cases, the designed complexes did not form, highlighting the challenges associated with designing buried polar interactions.

We computationally designed a de novo protein-protein interaction between wild-type ubiquitin and a redesigned scaffold. Our strategy was to incorporate zinc at the designed interface to promote affinity and orientation specificity. A large set of monomeric scaffold surfaces were computationally engineered with three-residue zinc coordination sites, and the ubiquitin residue H68 was docked to the open coordination site to complete a tetrahedral zinc site. This single coordination bond was intended as a hotspot and polar interaction for ubiquitin binding, and surrounding residues on the scaffold were optimized primarily as hydrophobic residues using a rotamer-based sequence design protocol in Rosetta. From thousands of independent design simulations, four sequences were selected for experimental characterization. The best performing design, called Spelter, binds tightly to zinc (Kd

Over the past decade, the Rosetta biomolecular modeling suite has informed diverse biological questions and engineering challenges ranging from interpretation of low-resolution structural data to design of nanomaterials, protein therapeutics, and vaccines. Central to Rosetta’s success is the energy function: amodel parameterized from small molecule and X-ray crystal structure data used to approximate the energy associated with each biomolecule conformation. This paper describes the mathematical models and physical concepts that underlie the latest Rosetta energy function, beta_nov15. Applying these concepts,we explain how to use Rosetta energies to identify and analyze the features of biomolecular models.Finally, we discuss the latest advances in the energy function that extend capabilities from soluble proteins to also include membrane proteins, peptides containing non-canonical amino acids, carbohydrates, nucleic acids, and other macromolecules.

The epigenetic inheritance of DNA methylation requires UHRF1, a histone- and DNA-binding RING E3 ubiquitin ligase that recruits DNMT1 to sites of newly replicated DNA through ubiquitylation of histone H3. UHRF1 binds DNA with selectivity towards hemi-methylated CpGs (HeDNA); however, the contribution of HeDNA sensing to UHRF1 function remains elusive. Here, we reveal that the interaction of UHRF1 with HeDNA is required for DNA methylation but is dispensable for chromatin interaction, which is governed by reciprocal positive cooperativity between the UHRF1 histone- and DNA-binding domains. HeDNA recognition activates UHRF1 ubiquitylation towards multiple lysines on the H3 tail adjacent to the UHRF1 histone-binding site. Collectively, our studies are the first demonstrations of a DNA-protein interaction and an epigenetic modification directly regulating E3 ubiquitin ligase activity. They also define an orchestrated epigenetic control mechanism involving modifications both to histones and DNA that facilitate UHRF1 chromatin targeting, H3 ubiquitylation, and DNA methylation inheritance. DOI: http://dx.doi.org/10.7554/eLife.17101.001, eLife digest Cells are able to regulate the activity of their genes in response to different cues. Genetic information is encoded in DNA and one way to regulate gene activity is to modify the DNA by attaching chemical “epigenetic” markers to it. When a cell divides, these epigenetic markers can be inherited by the daughter cells so that they share the same patterns of gene activity as the parent cell. When the DNA of the parent cell is copied prior to cell division, the epigenetic markers are also copied onto the new DNA. Mistakes in this process are linked to a wide range of diseases in humans, such as cancer and neurological disorders. One type of epigenetic marker is known as a methyl tag and it is added to DNA by certain enzymes in a process called DNA methylation. A protein called UHRF1 is required for human cells to inherit patterns of DNA methylation through cell division. This protein binds to newly copied DNA that lacks some methyl tags as well as to another protein associated with DNA called histone H3. UHRF1 modifies histone H3 by attaching a small protein molecule called ubiquitin to it. This helps to recruit a DNA methylation enzyme to place methyl tags on the newly copied DNA. However, it was not clear how the various properties of UHRF1 allow it to control how DNA methylation is inherited. Harrison et al. addressed this question by studying purified proteins and DNA fragments outside of living cells. The results show that UHRF1 binding to DNA and histone H3 work together to bring UHRF1 to the sites on DNA that require methylation. Further experiments revealed that the methylation pattern on newly copied DNA is able to activate the ability of UHRF1 to place ubiquitin on histone H3. The findings of Harrison et al. reveal a new mechanism by which dividing cells control how DNA methylation is inherited by their daughter cells. A future challenge will be to find out how attaching ubiquitin to histone H3 activates DNA methylation. DOI: http://dx.doi.org/10.7554/eLife.17101.002

Hypoxia-inducible factors (HIFs) are heterodimeric transcription factors central to hypoxia response and cancer development. Within the HIF-2 complex, one domain (HIF-2α PAS-B) contains a large (290 A3) buried cavity filled with water molecules within its hydrophobic core. Such cavities are uncommon except in the case of ligand-binding proteins, leading to the hypothesis that HIF-2α can be regulated by small molecules. The development of artificial HIF-2α inhibitors validates this hypothesis but raises questions about the impact of this cavity on HIF-2α PAS-B structure and function. To answer these points, we used computational methods to construct a repacked protein containing a smaller cavity within the native fold. Experimental validation of a five-mutation variant confirms achieving these objectives and stabilizing the folded structure. Complementary functional data establish that ligands cannot bind this variant although heterodimerization remains unchanged. Altogether, our strategy innovatively addresses the roles of solvated cavities in maintaining protein stability and function.

Haptotaxis is the process by which cells respond to gradients of substrate-bound cues, such as extracellular matrix proteins (ECM); however, the cellular mechanism of this response remains poorly understood and has mainly been studied by comparing cell behavior on uniform ECMs with different concentrations of components. To study haptotaxis in response to gradients, we utilized microfluidic chambers to generate gradients of the ECM protein fibronectin, and imaged the cell migration response. Lamellipodia are fan-shaped protrusions that are common in migrating cells. Here, we define a new function for lamellipodia and the cellular mechanism required for haptotaxis – differential actin and lamellipodial protrusion dynamics lead to biased cell migration. Modest differences in lamellipodial dynamics occurring over time periods of seconds to minutes are summed over hours to produce differential whole cell movement towards higher concentrations of fibronectin. We identify a specific subset of lamellipodia regulators as being crucial for haptotaxis. Numerous studies have linked components of this pathway to cancer metastasis and, consistent with this, we find that expression of the oncogenic Rac1 P29S mutation abrogates haptotaxis. Finally, we show that haptotaxis also operates through this pathway in 3D environments.

Structure-based protein design tests our understanding of the minimal determinants of protein structure and function. Previous studies have demonstrated that placing zinc binding amino acids (His, Glu, Asp or Cys) near each other in a folded protein in an arrangement predicted to be tetrahedral is often sufficient to achieve binding to zinc. However, few designs have been characterized with high-resolution structures. Here, we use X-ray crystallography, binding studies and mutation analysis to evaluate three alternative strategies for designing zinc binding sites with the molecular modeling program Rosetta. While several of the designs were observed to bind zinc, crystal structures of two designs reveal binding configurations that differ from the design model. In both cases, the modeling did not accurately capture the presence or absence of second-shell hydrogen bonds critical in determining binding-site structure. Efforts to more explicitly design second-shell hydrogen bonds were largely unsuccessful as evidenced by mutation analysis and low expression of proteins engineered with extensive primary and secondary networks. Our results suggest that improved methods for designing interaction networks will be needed for creating metal binding sites with high accuracy.

Naturally occurring proteins often consist of multiple distinct domains joined by linker regions. Similarly, the ability to combine globular protein domains through engineered linkers would allow the creation of a wide variety of complex and useful multifunctional proteins. Recent advances in computational design of protein structures have enabled highly accurate design of novel protein structures. In this chapter we outline a computational protocol for the de novo design of protein linkers, and apply this protocol to the design of a helical linker between two rigid protein domains.

Lysine selectivity is of critical importance during polyubiquitin chain formation because the identity of the lysine controls the biological outcome. Ubiquitins are covalently linked in polyubiquitin chains through one of seven lysine residues on its surface and the C terminus of adjacent protomers. Lys 48-linked polyubiquitin chains signal for protein degradation; however, the structural basis for Lys 48 selectivity remains largely unknown. The ubiquitin-conjugating enzyme Ube2R1/2 has exquisite specificity for Lys 48, and computational docking of Ube2R1/2 and ubiquitin predicts that Lys 48 is guided to the active site through a key electrostatic interaction between Arg 54 on ubiquitin and Asp 143 on Ube2R1/2. The validity of this interaction was confirmed through biochemical experiments. Since structural examples involving Arg 54 in protein-ubiquitin complexes are exceedingly rare, these results provide additional insight into how ubiquitin-protein complexes can be stabilized. We discuss how these findings relate to how other ubiquitin-conjugating enzymes direct the lysine specificity of polyubiquitin chains.

Cell growth and differentiation are controlled by growth factor receptors coupled to the GTPase Ras. Oncogenic mutations disrupt GTPase activity, leading to persistent Ras signaling and cancer progression. Recent evidence indicates that monoubiquitination of Ras leads to Ras activation. Mutation of the primary site of monoubiquitination impairs the ability of activated K-Ras (one of the three mammalian isoforms of Ras) to promote tumor growth. To determine the mechanism of human Ras activation, we chemically ubiquitinated the protein and analyzed its function by NMR, computational modeling and biochemical activity measurements. We established that monoubiquitination has little effect on the binding of Ras to guanine nucleotide, GTP hydrolysis or exchange-factor activation but severely abrogates the response to GTPase-activating proteins in a site-specific manner. These findings reveal a new mechanism by which Ras can trigger persistent signaling in the absence of receptor activation or an oncogenic mutation.

SummaryProtein design tests our understanding of protein stability and structure. Successful design methods should allow the exploration of sequence space not found in nature. However, when redesigning naturally occurring protein structures, most fixed backbone design algorithms return amino acid sequences that share strong sequence identity with wild-type sequences, especially in the protein core. This behavior places a restriction on functional space that can be explored and is not consistent with observations from nature, where sequences of low identity have similar structures. Here, we allow backbone flexibility during design to mutate every position in the core (38 residues) of a four-helix bundle protein. Only small perturbations to the backbone, 1–2 Å, were needed to entirely mutate the core. The redesigned protein, DRNN, is exceptionally stable (melting point >140°C). An NMR and X-ray crystal structure show that the side chains and backbone were accurately modeled (all-atom RMSD = 1.3 Å).

Pathways of ubiquitin-like (UBL) molecule transfer regulate a myriad of cellular cascades. Here, we report a high-throughput assay that correlates catalytic human NEDD8 transfer to bacterial survival. The assay was utilized to screen mutant NEDD8 and NAE (NEDD8-activating enzyme) libraries to engineer a more stable NEDD8 and redesign the NEDD8-NAE interface. This approach will be useful in understanding the specificities underlying UBL pathways.

Summary Mutation of surface residues to charged amino acids increases resistance to aggregation and can enable reversible unfolding. We have developed a protocol using the Rosetta computational design package that "supercharges" proteins while considering the energetic implications of each mutation. Using a homology model, a single-chain variable fragment antibody was designed that has a markedly enhanced resistance to thermal inactivation and displays an unanticipated ≈30-fold improvement in affinity. Such supercharged antibodies should prove useful for assays in resource-limited settings and for developing reagents with improved shelf lives.

Computational design of novel protein–protein interfaces is a test of our understanding of protein interactions and has the potential to allow modification of cellular physiology. Methods for designing high-affinity interactions that adopt a predetermined binding mode have proved elusive, suggesting the need for new strategies that simplify the design process. A solvent-exposed backbone on a β-strand is thought of as “sticky” and β-strand pairing stabilizes many naturally occurring protein complexes. Here, we computationally redesign a monomeric protein to form a symmetric homodimer by pairing exposed β-strands to form an intermolecular β-sheet. A crystal structure of the designed complex closely matches the computational model (rmsd = 1.0 Å ). This work demonstrates that β-strand pairing can be used to computationally design new interactions with high accuracy.

Fluorescent biosensors for living cells currently require laborious optimization and a unique design for each target. They are limited by the availability of naturally occurring ligands with appropriate target specificity. Here we describe a biosensor based on an engineered fibronectin monobody scaffold that can be tailored to bind different targets via high throughput screening. This Src family kinase (SFK) biosensor was made by derivatizing a monobody specific for activated SFK with a bright dye whose fluorescence increases upon target binding. We identified sites for dye attachment and alterations to eliminate vesiculation in living cells, providing a generalizable scaffold for biosensor production. This approach minimizes cell perturbation because it senses endogenous, unmodified target, and because sensitivity is enhanced by direct dye excitation. Automated correlation of cell velocities and SFK activity revealed that SFK are activated specifically during protrusion. Activity correlates with velocity, and peaks 1–2 microns from the leading edge.

We substantiate our preliminary description of the class I tryptophanyl-tRNA synthetase minimal catalytic domain with details of its construction, structure, and steady-state kinetic parameters. Generating that active fragment involved deleting 65% of the contemporary enzyme, including the anticodon-binding domain and connecting peptide 1, CP1, a 74-residue internal segment from within the Rossmann fold. We used protein design (Rosetta), rather than phylogenetic sequence alignments, to identify mutations to compensate for the severe loss of modularity, thus restoring stability, as evidenced by renaturation described previously and by 70-ns molecular dynamics simulations. Sufficient solubility to enable biochemical studies was achieved by expressing the redesigned Urzyme as a maltose-binding protein fusion. Michaelis-Menten kinetic parameters from amino acid activation assays showed that, compared with the native full-length enzyme, TrpRS Urzyme binds ATP with similar affinity. This suggests that neither of the two deleted structural modules has a strong influence on ground-state ATP binding. However, tryptophan has 103 lower affinity, and the Urzyme has comparably reduced specificity relative to the related amino acid, tyrosine. Molecular dynamics simulations revealed how CP1 may contribute significantly to cognate amino acid specificity. As class Ia editing domains are nested within the CP1, this finding suggests that this module enhanced amino acid specificity continuously, throughout their evolution. We call this type of reconstructed protein catalyst an Urzyme (Ur prefix indicates original, primitive, or earliest). It establishes a model for recapitulating very early steps in molecular evolution in which fitness may have been enhanced by accumulating entire modules, rather than by discrete amino acid sequence changes.

DNA methylation patterns regulate gene expression programs and are maintained through a highly coordinated process orchestrated by the RING E3 ubiquitin ligase UHRF1. UHRF1 controls DNA methylation inheritance by reading epigenetic modifications to histones and DNA to activate histone H3 ubiquitylation. Here, we find that all five domains of UHRF1, including the previously uncharacterized ubiquitin-like domain (UBL), cooperate for hemi-methylated DNA-dependent H3 ubiquitin ligation. Our structural and biochemical studies, including mutations found in cancer genomes, reveal a bifunctional requirement for the UBL in histone modification: (1) the UBL makes an essential interaction with the backside of the E2 and (2) the UBL coordinates with other UHRF1 domains that recognize epigenetic marks on DNA and histone H3 to direct ubiquitin to H3. Finally, we show UBLs from other E3s also have a conserved interaction with the E2, Ube2D, highlighting a potential prevalence of interactions between UBLs and E2s.

Computational algorithms for protein design can sample large regions of sequence space, but suffer from undersampling of conformational space and energy function inaccuracies. Experimental screening of combinatorial protein libraries avoids the need for accurate energy functions, but has limited access to vast amounts of sequence space. Here, we test if these two traditionally alternative, but potentially complementary approaches can be combined to design a variant of the ubiquitin-ligase E6AP that will bind to a nonnatural partner, the NEDD8-conjugating enzyme Ubc12. Three E6AP libraries were constructed: ( i ) a naive library in which all 20 amino acids were allowed at every position on the target-binding surface of E6AP (13 positions), ( ii ) a semidirected library that varied the same residue positions as in the naive library but disallowed mutations computationally predicted to destabilize E6AP, and ( iii ) a directed library that used docking and sequence optimization simulations to identify mutations predicted to be favorable for binding Ubc12. Both of the directed libraries showed > 30-fold enrichment over the naive library after the first round of screening with a split-dihydrofolate reductase complementation assay and produced multiple tight binders ( K d K d ’s lower than 50 μM. These results indicate that protein design simulations can be used to create directed libraries that are enriched in tight binders and that in some cases it is sufficient to computationally screen for well-folded sequences without explicit binding calculations.