Inside-Out Design of Zinc-Binding Proteins with Non-Native Backbones.

The de novo design of functional proteins requires specification of tertiary structure and incorporation of molecular binding sites. Here, we develop an inside-out design strategy in the molecular modeling program Rosetta that begins with amino acid side chains from one or two α-helices making well-defined contacts with a ligand. A full-sized protein is then built around the ligand by adding additional helices that promote the formation of a protein core and allow additional contacts with the ligand. The protocol was tested by designing 12 zinc-binding proteins, each with 4-5 helices. Four of the designs were folded and bound to zinc with equilibrium dissociation constants varying between 95 nM and 1.1 µM. The design with the tightest affinity for zinc, N12, adopts a unique conformation in the folded state as assessed with nuclear magnetic resonance (NMR) and the design model closely matches (backbone root-mean-square deviation (RMSD) < 1 Å) an AlphaFold model of the sequence. Retrospective analysis with AlphaFold suggests that the sequences of many of the failed designs did not encode the desired tertiary packing.

Targeted Mutagenesis of the Multicopy Myrosinase Gene Family in Allotetraploid Brassica juncea Reduces Pungency in Fresh Leaves across Environments.

Recent breeding efforts in Brassica have focused on the development of new oilseed feedstock crop for biofuels (e.g., ethanol, biodiesel, bio-jet fuel), bio-industrial uses (e.g., bio-plastics, lubricants), specialty fatty acids (e.g., erucic acid), and producing low glucosinolates levels for oilseed and feed meal production for animal consumption. We identified a novel opportunity to enhance the availability of nutritious, fresh leafy greens for human consumption. Here, we demonstrated the efficacy of disarming the 'mustard bomb' reaction in reducing pungency upon the mastication of fresh tissue-a major source of unpleasant flavor and/or odor in leafy Brassica. Using gene-specific mutagenesis via CRISPR-Cas12a, we created knockouts of all functional copies of the type-I myrosinase multigene family in tetraploid Brassica juncea. Our greenhouse and field trials demonstrate, via sensory and biochemical analyses, a stable reduction in pungency in edited plants across multiple environments. Collectively, these efforts provide a compelling path toward boosting the human consumption of nutrient-dense, fresh, leafy green vegetables.

Computationally designed peptide macrocycle inhibitors of New Delhi metallo-ß-lactamase 1.

The rise of antibiotic resistance calls for new therapeutics targeting resistance factors such as the New Delhi metallo-ß-lactamase 1 (NDM-1), a bacterial enzyme that degrades ß-lactam antibiotics. We present structure-guided computational methods for designing peptide macrocycles built from mixtures of l- and d-amino acids that are able to bind to and inhibit targets of therapeutic interest. Our methods explicitly consider the propensity of a peptide to favor a binding-competent conformation, which we found to predict rank order of experimentally observed IC50 values across seven designed NDM-1- inhibiting peptides. We were able to determine X-ray crystal structures of three of the designed inhibitors in complex with NDM-1, and in all three the conformation of the peptide is very close to the computationally designed model. In two of the three structures, the binding mode with NDM-1 is also very similar to the design model, while in the third, we observed an alternative binding mode likely arising from internal symmetry in the shape of the design combined with flexibility of the target. Although challenges remain in robustly predicting target backbone changes, binding mode, and the effects of mutations on binding affinity, our methods for designing ordered, binding-competent macrocycles should have broad applicability to a wide range of therapeutic targets.

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.

Evolution of a highly active and enantiospecific metalloenzyme from short peptides.

Primordial sequence signatures in modern proteins imply ancestral origins tracing back to simple peptides. Although short peptides seldom adopt unique folds, metal ions might have templated their assembly into higher-order structures in early evolution and imparted useful chemical reactivity. Recapitulating such a biogenetic scenario, we have combined design and laboratory evolution to transform a zinc-binding peptide into a globular enzyme capable of accelerating ester cleavage with exacting enantiospecificity and high catalytic efficiency (k cat/K M ~ 106 M-1 s-1). The simultaneous optimization of structure and function in a naïve peptide scaffold not only illustrates a plausible enzyme evolutionary pathway from the distant past to the present but also proffers exciting future opportunities for enzyme design and engineering.

Protocols for Requirement-Driven Protein Design in the Rosetta Modeling Program.

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.

An excited state underlies gene regulation of a transcriptional riboswitch.

Riboswitches control gene expression through ligand-dependent structural rearrangements of the sensing aptamer domain. However, we found that the Bacillus cereus fluoride riboswitch aptamer adopts identical tertiary structures in solution with and without ligand. Using chemical-exchange saturation transfer (CEST) NMR spectroscopy, we revealed that the structured ligand-free aptamer transiently accesses a low-populated (∼1%) and short-lived (∼3 ms) excited conformational state that unravels a conserved 'linchpin' base pair to signal transcription termination. Upon fluoride binding, this highly localized, fleeting process is allosterically suppressed, which activates transcription. We demonstrated that this mechanism confers effective fluoride-dependent gene activation over a wide range of transcription rates, which is essential for robust toxicity responses across diverse cellular conditions. These results unveil a novel switching mechanism that employs ligand-dependent suppression of an aptamer excited state to coordinate regulatory conformational transitions rather than adopting distinct aptamer ground-state tertiary architectures, exemplifying a new mode of ligand-dependent RNA regulation.

Probing the minimal determinants of zinc binding with computational protein design.

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.

A novel intracellular fibulin-1D variant binds to the cytoplasmic domain of integrin beta 1 subunit.

Fibulin-1 is a member of a growing family of proteins that includes eight members and is involved in cellular functions such as adhesion, migration and differentiation. Fibulin-1 has also been implicated in embryonic development of the heart and neural crest-derived structures. It is an integral part of the extracellular matrix (ECM) and has been shown to bind to a multitude of ECM proteins. However, fibulin-1 was first identified as a protein purified from placental extracts that binds to the cytoplasmic domain of integrin ß1. Human fibulin-1 is alternatively spliced into four different isoforms namely A-D. These isoforms share a common N-terminus sequence that contains a secretion sequence but differ in their carboxy-terminal fibulin-1 module. In this report we identify a new splice variant of fibulin-1 that differs from all other fibulin-1 variants in the N-terminus sequence and has a similar carboxy-terminus sequence as fibulin-1D. This variant that we named fibulin-1D prime (fibulin-1D') lacks a secretion sequence and the anaphlatoxin region of fibulin-1 variants. The protein has an apparent molecular weight of 70.5kDa. Herein we show that fibulin-1D' binds to the intracellular domain of integrin ß1 as well as to integrin α5ß1. The protein was localized intracellularly in CHO cells transfected with a pEF4 plasmid containing full-length coding sequence of fibulin-1D'. We also localized the protein in human placenta. We propose that the fibulin-1D' variant might play a role in early embryo development as well as in modulating integrin ß1 functions including adhesion and motility.