De novo design of buttressed loops for sculpting protein functions.

In natural proteins, structured loops have central roles in molecular recognition, signal transduction and enzyme catalysis. However, because of the intrinsic flexibility and irregularity of loop regions, organizing multiple structured loops at protein functional sites has been very difficult to achieve by de novo protein design. Here we describe a solution to this problem that designs tandem repeat proteins with structured loops (9-14 residues) buttressed by extensive hydrogen bonding interactions. Experimental characterization shows that the designs are monodisperse, highly soluble, folded and thermally stable. Crystal structures are in close agreement with the design models, with the loops structured and buttressed as designed. We demonstrate the functionality afforded by loop buttressing by designing and characterizing binders for extended peptides in which the loops form one side of an extended binding pocket. The ability to design multiple structured loops should contribute generally to efforts to design new protein functions.

De novo design of buttressed loops for sculpting protein functions.

In natural proteins, structured loops play central roles in molecular recognition, signal transduction and enzyme catalysis. However, because of the intrinsic flexibility and irregularity of loop regions, organizing multiple structured loops at protein functional sites has been very difficult to achieve by de novo protein design. Here we describe a solution to this problem that generates structured loops buttressed by extensive hydrogen bonding interactions with two neighboring loops and with secondary structure elements. We use this approach to design tandem repeat proteins with buttressed loops ranging from 9 to 14 residues in length. Experimental characterization shows the designs are folded and monodisperse, highly soluble, and thermally stable. Crystal structures are in close agreement with the computational design models, with the loops structured and buttressed by their neighbors as designed. We demonstrate the functionality afforded by loop buttressing by designing and characterizing binders for extended peptides in which the loops form one side of an extended binding pocket. The ability to design multiple structured loops should contribute quite generally to efforts to design new protein functions.

Deploying synthetic coevolution and machine learning to engineer protein-protein interactions.

Fine-tuning of protein-protein interactions occurs naturally through coevolution, but this process is difficult to recapitulate in the laboratory. We describe a platform for synthetic protein-protein coevolution that can isolate matched pairs of interacting muteins from complex libraries. This large dataset of coevolved complexes drove a systems-level analysis of molecular recognition between Z domain-affibody pairs spanning a wide range of structures, affinities, cross-reactivities, and orthogonalities, and captured a broad spectrum of coevolutionary networks. Furthermore, we harnessed pretrained protein language models to expand, in silico, the amino acid diversity of our coevolution screen, predicting remodeled interfaces beyond the reach of the experimental library. The integration of these approaches provides a means of simulating protein coevolution and generating protein complexes with diverse molecular recognition properties for biotechnology and synthetic biology.

Design of protein-binding proteins from the target structure alone.

The design of proteins that bind to a specific site on the surface of a target protein using no information other than the three-dimensional structure of the target remains a challenge1-5. Here we describe a general solution to this problem that starts with a broad exploration of the vast space of possible binding modes to a selected region of a protein surface, and then intensifies the search in the vicinity of the most promising binding modes. We demonstrate the broad applicability of this approach through the de novo design of binding proteins to 12 diverse protein targets with different shapes and surface properties. Biophysical characterization shows that the binders, which are all smaller than 65 amino acids, are hyperstable and, following experimental optimization, bind their targets with nanomolar to picomolar affinities. We succeeded in solving crystal structures of five of the binder-target complexes, and all five closely match the corresponding computational design models. Experimental data on nearly half a million computational designs and hundreds of thousands of point mutants provide detailed feedback on the strengths and limitations of the method and of our current understanding of protein-protein interactions, and should guide improvements of both. Our approach enables the targeted design of binders to sites of interest on a wide variety of proteins for therapeutic and diagnostic applications.

Glucose-dependent control of leucine metabolism by leucyl-tRNA synthetase 1.

Despite the importance of glucose and amino acids for energy metabolism, interactions between the two nutrients are not well understood. We provide evidence for a role of leucyl-tRNA synthetase 1 (LARS1) in glucose-dependent control of leucine usage. Upon glucose starvation, LARS1 was phosphorylated by Unc-51 like autophagy activating kinase 1 (ULK1) at the residues crucial for leucine binding. The phosphorylated LARS1 showed decreased leucine binding, which may inhibit protein synthesis and help save energy. Leucine that is not used for anabolic processes may be available for catabolic pathway energy generation. The LARS1-mediated changes in leucine utilization might help support cell survival under glucose deprivation. Thus, depending on glucose availability, LARS1 may help regulate whether leucine is used for protein synthesis or energy production.

MSK1 functions as a transcriptional coactivator of p53 in the regulation of p21 gene expression.

Mitogen- and stress-activated kinase 1 (MSK1) is a chromatin kinase that facilitates activator-dependent transcription by altering chromatin structure through histone H3 phosphorylation. The kinase activity of MSK1 is activated by intramolecular autophosphorylation, which is initially triggered by the activation of upstream mitogen-activated protein kinases (MAPKs), such as p38 and ERK1/2. MSK1 has been implicated in the expression of p21, a p53 target gene; however, the precise connection between MSK1 and p53 has not been clearly elucidated. Here, using in vitro and cell-based transcription assays, we show that MSK1 functions as a transcriptional coactivator of p53 in p21 expression, an action associated with MAPK-dependent phosphorylation of MSK1 and elevated kinase activity. Of special significance, we show that MSK1 directly interacts with p53 and is recruited to the p21 promoter, where it phosphorylates histone H3 in a p53-dependent manner. In addition, phosphomimetic mutant analysis demonstrated that negative charges in the hydrophobic motif are critical for serine 212 phosphorylation in the N-terminal kinase domain, which renders MSK1 competent for histone kinase activity. These studies suggest that MSK1 acts through a direct interaction with p53 to function as a transcriptional coactivator and that MSK1 activation by upstream MAPK signaling is important for efficient p21 gene expression.

Chemical biology approaches for studying posttranslational modifications.

Posttranslational modification (PTM) is a key mechanism for regulating diverse protein functions, and thus critically affects many essential biological processes. Critical for systematic study of the effects of PTMs is the ability to obtain recombinant proteins with defined and homogenous modifications. To this end, various synthetic and chemical biology approaches, including genetic code expansion and protein chemical modification methods, have been developed. These methods have proven effective for generating site-specific authentic modifications or structural mimics, and have demonstrated their value for in vitro and in vivo functional studies of diverse PTMs. This review will discuss recent advances in chemical biology strategies and their application to various PTM studies.

Expanding the genetic code of Mus musculus.

Here we report the expansion of the genetic code of Mus musculus with various unnatural amino acids including Nɛ-acetyl-lysine. Stable integration of transgenes encoding an engineered Nɛ-acetyl-lysyl-tRNA synthetase (AcKRS)/tRNAPyl pair into the mouse genome enables site-specific incorporation of unnatural amino acids into a target protein in response to the amber codon. We demonstrate temporal and spatial control of protein acetylation in various organs of the transgenic mouse using a recombinant green fluorescent protein (GFPuv) as a model protein. This strategy will provide a powerful tool for systematic in vivo study of cellular proteins in the most commonly used mammalian model organism for human physiology and disease.

A chemical biology route to site-specific authentic protein modifications.

Many essential biological processes are controlled by posttranslational protein modifications. The inability to synthetically attain the diversity enabled by these modifications limits functional studies of many proteins. We designed a three-step approach for installing authentic posttranslational modifications in recombinant proteins. We first use the established O-phosphoserine (Sep) orthogonal translation system to create a Sep-containing recombinant protein. The Sep residue is then dephosphorylated to dehydroalanine (Dha). Last, conjugate addition of alkyl iodides to Dha, promoted by zinc and copper, enables chemoselective carbon-carbon bond formation. To validate our approach, we produced histone H3, ubiquitin, and green fluorescent protein variants with site-specific modifications, including different methylations of H3K79. The methylated histones stimulate transcription through histone acetylation. This approach offers a powerful tool to engineer diverse designer proteins.

Chromatin Kinases Act on Transcription Factors and Histone Tails in Regulation of Inducible Transcription.

The inflammatory response requires coordinated activation of both transcription factors and chromatin to induce transcription for defense against pathogens and environmental insults. We sought to elucidate the connections between inflammatory signaling pathways and chromatin through genomic footprinting of kinase activity and unbiased identification of prominent histone phosphorylation events. We identified H3 serine 28 phosphorylation (H3S28ph) as the principal stimulation-dependent histone modification and observed its enrichment at induced genes in mouse macrophages stimulated with bacterial lipopolysaccharide. Using pharmacological and genetic approaches, we identified mitogen- and stress-activated protein kinases (MSKs) as primary mediators of H3S28ph in macrophages. Cell-free transcription assays demonstrated that H3S28ph directly promotes p300/CBP-dependent transcription. Further, MSKs can activate both signal-responsive transcription factors and the chromatin template with additive effects on transcription. Specific inhibition of MSKs in macrophages selectively reduced transcription of stimulation-induced genes. Our results suggest that MSKs incorporate upstream signaling inputs and control multiple downstream regulators of inducible transcription.

Akt Kinase-Mediated Checkpoint of cGAS DNA Sensing Pathway.

Upon DNA stimulation, cyclic GMP-AMP synthase (cGAS) synthesizes the second messenger cyclic GMP-AMP (cGAMP) that binds to the STING, triggering antiviral interferon-ß (IFN-ß) production. However, it has remained undetermined how hosts regulate cGAS enzymatic activity after the resolution of DNA immunogen. Here, we show that Akt kinase plays a negative role in cGAS-mediated anti-viral immune response. Akt phosphorylated the S291 or S305 residue of the enzymatic domain of mouse or human cGAS, respectively, and this phosphorylation robustly suppressed its enzymatic activity. Consequently, expression of activated Akt led to the reduction of cGAMP and IFN-ß production and the increase of herpes simplex virus 1 replication, whereas treatment with Akt inhibitor augmented cGAS-mediated IFN-ß production. Furthermore, expression of the phosphorylation-resistant cGAS S291A mutant enhanced IFN-ß production upon DNA stimulation, HSV-1 infection, and vaccinia virus infection. Our study identifies an Akt kinase-mediated checkpoint to fine-tune hosts' immune responses to DNA stimulation.

Negative regulation of NF-κB activity by brain-specific TRIpartite Motif protein 9.

The TRIpartite Motif (TRIM) family of RING-domain-containing proteins participate in a variety of cellular functions. The ß-transducin repeat-containing protein (ß-TrCP), a component of the Skp-Cullin-F-box-containing (SCF) E3 ubiquitin ligase complex, recognizes the NF-κB inhibitor IκBα and precursor p100 for proteasomal degradation and processing, respectively. ß-TrCP thus plays a critical role in both canonical and non-canonical NF-κB activation. Here we report that TRIM9 is a negative regulator of NF-κB activation. Interaction between the phosphorylated degron motif of TRIM9 and the WD40 repeat region of ß-TrCP prevented ß-TrCP from binding its substrates, stabilizing IκBα and p100 and thereby blocking NF-κB activation. Consequently, expression or depletion of the TRIM9 gene significantly affected NF-κB-induced inflammatory cytokine production. This study not only elucidates a mechanism for TRIM9-mediated regulation of the ß-TrCP SCF complex activity but also identifies TRIM9 as a brain-specific negative regulator of the NF-κB pro-inflammatory signalling pathway.

Facile "stop codon" method reveals elevated neuronal toxicity by discrete S87p-α-synuclein oligomers.

Herein, a new method for preparing phosphorylated proteins at specific sites has been applied to α-synuclein (α-Syn). Three different α-Syn species phosphorylated at Serine 87 (S87p-α-Syn), Serine 129 (S129p-α-Syn) and Serine 87/129 (S87p,129p-α-Syn) were prepared through the 'stop codon' method and verified by LC/MS/MS and immunoblotting. Each type of phosphorylated α-Syn was tested for oligomerization trends and cellular toxicity with dopamine (DA), Cu(2+) ions and pyridoxal 5'-phosphate. Aggregation trends induced by DA or DA/Cu(2+) were similar between phosphorylated and non-phosphorylated α-Syn in SDS-PAGE. However, except for the monomer, phosphorylated oligomers showed higher toxicity than the non-phosphorylated α-Syn (Np-α-Syn) oligomers via WST-1 assays when tested on SH-SY5Y human neuroblastoma cells. In particular, S87p-α-Syn and S87p,129p-α-Syn oligomers induced by DA/Cu(2+), showed higher toxicity than did S129p-α-Syn. When α-Syn was treated with pyridoxal 5'-phosphate in the presence of DA or Cu(2+) to determine aggregation effects, high inhibition effects were shown in both non-phosphorylated and phosphorylated versions. α-Syn co-incubated with DA or DA/Cu(2+) showed less cellular toxicity upon pyridoxal 5'-phosphate treatment, especially in the case of DA-induced Np-α-syn. This study supports that phosphorylated oligomers of α-Syn at residue 87 can contribute to neuronal toxicity and the pyridoxal 5'-phosphate can be used as an inhibitor for α-Syn aggregation.

Dopamine and Cu+/2+ can induce oligomerization of α-synuclein in the absence of oxygen: Two types of oligomerization mechanisms for α-synuclein and related cell toxicity studies.

α-Synuclein oligomers can induce neurotoxicity and are implicated in Parkinson's disease etiology and disease progression. Many studies have reported α-synuclein oligomerization by dopamine (DA) and transition metal ions, but few studies provide insight into joint influences of DA and Cu2+ . In this study, DA and Cu2+ were coadministered aerobically to measure α-synuclein oligomerization under these conditions. In the presence of oxygen, DA induced α-synuclein oligomerization in a dose-dependent manner. Cu+/2+ did not effect oligomerization in such a manner in the presence of DA. By electrophoresis, Cu2+ was found easily to induce oligomerization with DA. This implies that oligomerization invoked by DA is reversible in the presence of Cu2+, which appears to be mediated by noncovalent bond interactions. In the absence of oxygen, DA induced less oligomerization of α-synuclein, whereas DA/Cu2+ induced aerobic-level amounts of oligomers, suggesting that DA/Cu2+ induces oligomerization independent of oxygen concentration. Radical species were detected through electron paramagnetic resonance (EPR) spectroscopic analysis arising from coincubation of DA/Cu2+ with α-synuclein. Redox reactions induced by DA/Cu2+ were observed in multimer regions of α-synuclein oligomers through NBT assay. Cellular toxicity results confirm that, for normal and hypoxic conditions, copper or DA/Cu2+ can induce cell death, which may arise from copper redox chemistry. From these results, we propose that DA and DA/Cu2+ induce different mechanisms of α-synuclein oligomerization, cross-linking with noncovalent (or reversible covalent) bonding vs. likely radical-mediated covalent modification.

Simple and efficient strategy for site-specific dual labeling of proteins for single-molecule fluorescence resonance energy transfer analysis.

Analysis of protein dynamics using single-molecule fluorescence resonance energy transfer (smFRET) is widely used to understand the structure and function of proteins. Nonetheless, site-specific labeling of proteins with a pair of donor and acceptor dyes still remains a challenge. Here we present a general and facile method for site-specific dual labeling of proteins by incorporating two different, readily available, unnatural amino acids (p-acetylphenylalanine and alkynyllysine) for smFRET. We used newly evolved alkynyllysine-specific aminoacyl-tRNA synthetase/tRNA(UCA) and p-acetylphenylalanyl-tRNA synthetase/tRNA(CUA). The utility of our approach was demonstrated by analyzing the conformational change of dual-labeled calmodulin using smFRET measurements. The present labeling approach is devoid of major limitations in conventional cysteine-based labeling. Therefore, our method will significantly increase the repertoire of proteins available for FRET study and expand our ability to explore more complicated molecular dynamics.