Angewandte Chemie International Edition

Activating KRAS mutations drive millions of cancers diagnosed worldwide,1 yet for decades this oncoprotein was deemed "undruggable", reflecting the challenge of discovering molecules capable of perturbing its complex biological functions, and of translating these discoveries into effective cancer therapeutics.2 Recent advances propelled by innovative screening have identified diverse modalities that bind at or near the switch-II pocket (SII-P) of RAS proteins, including molecular glues,3 macrocyclic peptides,4 fragment-derived small molecules,5 and approved therapies that covalently target KRASG12C.6,7 Unfortunately, resistance to approved therapies has emerged,8,9 highlighting the need for molecules that engage new or underexploited binding sites on RAS oncoproteins with mechanisms complementary to established SII-P inhibitors.10,11 Here we show that mirror-image mRNA display12 enabled the discovery of all-D macrocyclic peptide ligands targeting a cryptic RAS back pocket (CRB-P).13 These ligands engage KRAS(OFF) and KRAS(ON) with equal affinity, exploit a single-residue difference among isoforms to bind KRAS selectively, and successfully inhibit oncogenic signaling in KRAS-mutant cells through a mechanism distinct from SII-P binders. Mirror-image screening directly afforded nanomolar peptide ligands stable toward cellular proteolysis and delivered probes targeting distinct epitopes not accessible by homochiral peptide-display methods. Together, these findings establish the CRB-P as a specifically druggable and mechanistically differentiated site on KRAS with potential for combination with emerging RAS-targeting therapies and substantiate mirror-image mRNA display as a strategy for discovering stable all-D macrocyclic peptides targeting previously inaccessible epitopes on challenging targets.

Guanine Exchange Factors (GEF) of the Dbl family are the main activators of RhoA GTPases. GEF and GTPase activity is tightly regulated at the subcellular level with fast kinetics. Therefore, to fully understand the function of Dbl GEFs requires their study in living cells. Towards developing molecular tools that reversibly and rapidly modulate the activity of endogenous GEFs in living cells, here we developed a general platform for engineering inhibitors against members of the Dbl family of GEFs using generative protein design. Engineered proteins showed high affinity and remarkable specificity for the target GEFs and modulated GEF activity both in vitro and in cells. In a proof-of-principle example, a GEF inhibitor was coupled to a light-activated module, enabling the optogenetic control of its activity in cells. These findings show that generative protein design can create modulators of intracellular signaling and broaden the range of tools available for biological research.

Photoswitchable ligands enable photocontrol of biomolecular activity by binding to targets in an isomer-dependent, light-responsive manner. Recent developments in ionizable photoswitchable ligands greatly expand their applications but introduce a major design challenge: light-responsive binding can depend on isomeric form, chemical substitution, and binding-induced shifts in protonation equilibria. These effects are tightly coupled, subtle in magnitude, and difficult to predict. Consequently, few computational methods have been developed and systematically benchmarked for quantitatively predicting them. Here, we establish a multiscale free-energy method and benchmark it against experimental data for a series of recently developed photoswitchable inhibitors of Escherichia coli dihydrofolate reductase (eDHFR), a crucial target in photopharmacology. Constant pH replica-exchange molecular dynamics and quantum mechanics/molecular mechanics umbrella sampling quantitatively characterize the ligands protonation-state change upon binding to the eDHFR active site. Thermodynamic integration simulations using alternative alchemical pathways, thermodynamic cycles, and protonation-state assignments were evaluated for predicting light-responsive affinity differentials and substituent effects. Direct cis-to-trans transformations with explicit treatment of environment-dependent protonation states best reproduce experimental trends. Compound-to-compound pathways are less reliable because force-field inaccuracies introduce large pK errors that are difficult to correct when protonation/deprotonation processes implicitly enter the thermodynamic cycle. TI simulations that ignore binding-induced protonation-state changes fail to consistently reproduce experimental trends. Protein-ligand and ligand-water interaction analyses further reveal the energetic and structural origins of isomer-dependent binding. This study establishes a systematic free-energy method for designing ionizable photoswitches in photopharmacology.

In Anabaena variabilis (Trichormus variabilis) phenylalanine ammonia-lyase (AvPAL), a conserved lid-like loop sits over the active site and has been studied both for its role in positioning a catalytic tyrosine and for its contribution to phenylalanine aminomutase (PAM) activity. While the active site architecture and substrate specificity of AvPAL have been extensively characterized, the dynamic behavior of this unstructured loop beyond its role in catalysis remains poorly understood. Here, we investigate the functional role of this loop by restricting its mobility through targeted interchain disulfide bond engineering. Three in-house approaches were designed to predict ideal cysteine residue pairs: (i) quantifying pair interaction energies via electrostatic and van der Waals forces, (ii) generating a contact map of residues within 5 [A] proximity, and (iii) implementing a machine-learning model trained on datasets from PDBCYS, SPX, and an internal database to rank cysteine pair likelihood within disulfide bond geometric constraints. Our machine-learning-guided strategy yielded a successful variant with complete oxidation efficiency in E. coli. Rigidification of this loop reveals that it also functions as a regulator of substrate specificity. Multi-scale molecular simulation analyses (molecular dynamics, metadynamics, quantum/molecular mechanics) reveal that this modification alters the active-site pocket by reducing the conformational dynamics of substrate binding. Our findings underscore the delicate balance between enzyme flexibility and catalytic efficiency, providing novel insights into the role of this understudied dynamic loop region in AvPAL.

Despite their therapeutic potential across a wide range of central nervous system (CNS) disorders, nucleic acid-based therapeutics are limited by inefficient delivery to deep brain regions at clinically viable doses. Transferrin receptor 1 (TfR1) has emerged as an attractive target for receptor-mediated transcytosis across the blood-brain barrier (BBB), enabling systemic delivery of biologics such as lysosomal enzymes and monoclonal antibodies. In this study, we demonstrated the translational potential of recently described TfR1-targeting camelid-derived single-domain antibodies (VHHs) for CNS delivery of siRNAs. When conjugated 1:1 to different tool siRNAs, these VHHs promote rapid and robust intracellular uptake, resulting in potent RNAi activity at low nanomolar concentrations in neural cells. Systemic administration of VHH-siRNA conjugates in wild-type mice, hTfR1 transgenic-mice and non-human primates revealed a favourable pharmacokinetic profile characterized by rapid TfR-dependent distributional clearance and efficient functional uptake in deep brain structures. This translated into durable target knockdown of 50-80% at both mRNA and protein levels and with ED50 below 1 mg/kg siRNA. Collectively, these findings establish our TfR1 targeting VHHs as a fit-for-purpose platform for the systemic delivery of therapeutic oligonucleotides to deep brain structures at clinically relevant doses, opening new avenues for the treatment of diverse CNS disorders. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/726486v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@13668eorg.highwire.dtl.DTLVardef@1b1feeeorg.highwire.dtl.DTLVardef@d7be2dorg.highwire.dtl.DTLVardef@6b221_HPS_FORMAT_FIGEXP M_FIG C_FIG

Antibody and protein-drug conjugates (XDCs) have emerged as promising cancer therapeutics, yet their clinical utility remains constrained by dose-limiting toxicities and narrow therapeutic windows. These safety challenges stem primarily from two factors: premature payload release during systemic circulation, and poor physicochemical properties inherent to the hydrophobic cytotoxic drugs they carry. Prior strategies attempted to address these limitations by appending water-soluble tags to reduce overall conjugate hydrophobicity, but achieved only modest improvements. As a result, the hydrophobic nature of cytotoxic payloads has remained a persistent obstacle in XDC development. Here, we report a fundamentally different chemical strategy that reframes this liability as a design opportunity. Rather than masking drug hydrophobicity, we exploit it as the driving force for self-assembly of facially amphiphilic protein-drug conjugates with programmable drug moieties (PDCs). In this architecture, the hydrophobic cytotoxic drug and the hydrophilic protein serve as the core and shell, respectively, spontaneously assembling into monodisperse, well-defined spherical protein nanotherapeutics of controlled size. This design principle transforms a longstanding physicochemical challenge into a functional engineering tool, enabling precise nanostructure formation without sacrificing potency. In vitro studies confirm that the resulting nanotherapeutics effectively kill cancer cells, establishing a strong foundation for further therapeutic development.

Arylmalonate decarboxylase (AMDase) stereoselectively converts disubstituted malonates to chiral carboxylic acids, but its substrate spectrum is very limited regarding the size of the smaller substituent. Inspired by the observation that (S)-selective AMDase variants also convert larger substrates, we unlocked the synthesis of the (R)-enantiomers of -aryl and -alkenyl n-butanoic and n-pentanoic acids, respectively, in exquisite enantiopurity.

Whole-tissue spatial proteomics level provides critical insights into region-specific biological regulations but remains challenging. Previously, we introduced the Micro-scaffold Assisted Spatial Proteomics (MASP) concept for whole-tissue mapping. However, this prototype required substantial development in spatial resolution, practicality, and throughput for practical application. Here we present a next-generation MASP technique (hex-MASP) featuring i) a new design of hexagonal-micro-wells fabricated with optimized Projection Micro-Stereolithography (P{micro}SL) 3D-printing, achieving high spatial resolution, sampling robustness and mechanical strength for reproducibly compartmentalizing even tough tissues; ii) enhanced throughput/effectiveness in sample preparation and LC-MS analysis with high quantitative quality. Applied to mouse brain, hex-MASP for the first time achieved in-depth, whole-tissue mapping for >6,000 proteins in mouse brains, with high spatial accuracy and excellent data quality. The substantially improved resolution revealed critical regional details across the entire brain, that were not previously captured, enabling precise depiction of protein distribution heterogeneity. This technique enabled the discovery of many unreported regionally-enriched proteins across brain structures. We further applied hex-MASP to investigate the intra-brain distribution of intracerebroventricularly-dosed antibody therapeutics and related proteins, which to our knowledge, enabled whole tissue mapping of protein drugs for the first time and revealed novel mechanistic insights into antibody distribution and localized treatment effects. Hex-MASP represent a robust, scalable platform for whole-tissue spatial proteomics.

Cytochrome P450s catalyze a diverse array of reactions including crosslinking of aromatic side chains in the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs). ApyO is a cytochrome P450 enzyme that forms a C-C bond between two tyrosines in a YLY motif in the substrate ApyA, the precursor peptide of the RiPP aminopyruvatide. We utilized cell-free translation to generate ApyA variants and probe the substrate tolerance of ApyO. Through Alphafold-based modelling and in vitro assays, we show that ApyO accepts the 10 C-terminal residues of ApyA and requires a conserved Arg/Lys in the substrate peptide. Inspired by substrate sequences found in orthologous biosynthetic gene clusters, we substituted one of the tyrosine residues with a tryptophan and observed that ApyO catalyzed the formation of an N-C bond between the indole of Trp and the C{varepsilon}2 of Tyr. ApyO unexpectedly catalyzed formation of a C-O bond between the two tyrosine residues when we substituted the leucine residue in the YLY motif with tyrosine and tryptophan. We also show that a peptide containing a biaryl linkage and the C-terminal aminopyruvate displayed sub-nanomolar inhibitory activity against selected proteases. Overall, this study demonstrates plasticity in the manner of macrocyclization catalyzed by the P450 ApyO and provides a starting point for chemoenzymatic approaches towards producing diverse macrocyclic scaffolds.

Despite intensive efforts, the ferroptosis gatekeeper glutathione peroxidase 4 (GPX4) remains difficult to selectively target due to stringent structural constraints surrounding its catalytic selenocysteine, which impose tight requirements on warhead reactivity and geometry. Here, leveraging a chemoproteomic approach, we characterize a potent and selective covalent GPX4 inhibitor featuring a pyrimidinylmethyl isourea warhead and define the chemical features underlying its proteome-wide selectivity. This chemotype enables tunable electrophile reactivity through steric and electronic modulation of leaving group ability, suggesting potential broader utility for targeting other recalcitrant proteins. Building on this scaffold, we further develop two selective GPX4 degraders - one CRBN-dependent and the other CRBN-independent - enabling complementary modulation of GPX4 through both inhibition and degradation. Together, these molecules expand the GPX4 chemical toolbox for more nuanced interrogation of GPX4 biology.

The use of covalent warheads targeting the catalytic cysteine has been a cornerstone in coronavirus main protease (Mpro) inhibitor development, where various electrophilic motifs have been used including aldehydes, nitriles, ketoamides, and hydroxymethyl ketones (HMKs). Recent efforts have been mostly centered around nitrile warheads, given the success of compounds like Nirmatrelvir and Ensitrelvir in the clinic. However, finding and advancing alternative chemotypes with differentiating chemical and pharmacological profiles is essential for future pandemic preparedness. Among such alternatives, HMKs hold special interest because they balance reduced intrinsic electrophilicity with an excellent selectivity profile. Nevertheless, early HMK-based compounds, such as the clinical-stage Mpro inhibitor PF-00835231, suffered from poor oral bioavailability and therefore required intravenous administration, with or without prodrug derivatization of the hydroxyl group. Here, we describe our efforts in advancing the HMK field via the discovery of mCMX110, a lead that has superior potency, increased unbound exposure in vivo, and favorable oral bioavailability in preclinical studies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/725542v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@abe1c9org.highwire.dtl.DTLVardef@746a08org.highwire.dtl.DTLVardef@dd5861org.highwire.dtl.DTLVardef@1d572c7_HPS_FORMAT_FIGEXP M_FIG C_FIG

Proton-sponge-active polymers are widely used in nanomedicine to enhance intracellular delivery, yet the mechanism by which they promote cytosolic release of therapeutic cargo remains under debate. Whether these materials drive complete endolysosomal escape or instead alter lysosomal integrity without full nanoparticle release remains unclear. Here we show that polyethylene imine (PEI), a prototypical proton sponge active polymer, induces lysosomal membrane destabilization rather than full nanoparticle escape. Using PEI-coated mesoporous silica nanoparticles as a model delivery system, we show that PEI promotes cytosolic release of small-molecule cargo while nanoparticles remain confined within membrane-enclosed LAMP1-positive compartments. This behaviour arises from the combination of partial lysosomal membrane permeabilization and lysosomal deacidification, which together enable cargo leakage while impairing detection of lysosomes by pH-dependent probes. Our results resolve a long-standing ambiguity in the nanomedicine field and provide a revised mechanistic framework for interpreting endolysosomal escape in intracellular delivery. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/721565v2_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@74b98org.highwire.dtl.DTLVardef@f405eborg.highwire.dtl.DTLVardef@b0a276org.highwire.dtl.DTLVardef@79f154_HPS_FORMAT_FIGEXP M_FIG C_FIG

The self-labeling protein HaloTag is used to install a wide variety of functional small molecules in cells and living organisms with exquisite specificity with respect to cell type and subcellular localization. HaloTag is a core part of many biotechnology-based tools for sensing, tracking, and manipulating biological systems with a high degree of spatial and temporal control. Due to the limitations of fluorescent proteins and other self-labeling proteins, most of these tools have historically been restricted to a single channel. In this work, we used structure-guided rational design and directed evolution to produce an orthogonal HaloTag protein called OrthoTag which reacts selectively with a modified chloroalkane substrate. OrthoTag retains many of HaloTags superior properties, and reaction rate measurements show OrthoTag and its substrate have 60-fold mutual orthogonality to HaloTag. We demonstrate the application of OrthoTag for multiplexed labeling experiments in mammalian cells with minimal optimization. Going forward, OrthoTag can be directly incorporated into any HaloTag-based system to allow simultaneous measurement or manipulation of two biological targets or processes. The availability of multiple high-performance self-labeling proteins will enable the continued development of new multiplexed biotechnology methods.

Genetic code expansion introduces new-to-nature chemical moieties into ribosomally synthesized proteins. In practice, the scope of functional groups that can be accessed using this method is often limited by noncanonical amino acid (ncAA) availability. Producing ncAAs directly in cells can circumvent poor ncAA uptake or commercial unavailability, but limited enzymes suitable for this application exist. In vitro evolution campaigns have been remarkably successful in yielding synthetically useful "ncAA synthases." However, these enzymes are optimized for preparative-scale synthesis and their activities often do not translate well to cellular biosynthesis. Thus, expanding strategies to engineer enzymes specifically for ncAA production within cells will benefit further implementation of genetic code expansion. Here, we use phage-assisted noncontinuous and continuous evolution to evolve enzymes for improved synthesis of non-canonical tyrosine derivatives in E. coli. Using simple serial passaging, we uncovered mutations that doubled the production of an expensive ncAA, 3-methoxytyrosine, by tyrosine phenol lyase, and furthermore evolved variants that enable 3-iodotyrosine biosynthesis, a transformation the parent enzyme is unable to catalyze. Additionally, we evolved a recently reported tyrosine synthase for improved production of 3-halogenated tyrosines, identifying variants that exhibit high activity even at low substrate concentrations owing to a [~]8-fold reduction in KM. Our results demonstrate that phage assisted evolution can be used to rapidly improve the activity of enzymes for ncAA production in cells.

Class B1 G-protein-coupled receptors (GPCRs), such as the calcitonin gene-related peptide (CGRP) receptor and parathyroid hormone 1 (PTH1) receptor, require native lipid interactions to maintain signalling-competent conformations. However, conventional detergents disrupt these environments. Amphipathic copolymers offer a detergent-free alternative, yet the field still lacks a clear understanding of which polymer architectures best preserve active-state GPCR pharmacology, limiting their broader translational utility. Here, we examine how distinct copolymer chemistries influence the functional integrity of class B1 GPCRs by comparing SMA 2000, DIBMA-12, and the electroneutral sulfo-DIBMA. Using NanoLuciferase bioluminescence resonance energy transfer (NanoBRET) ligand-binding, competition, and mini-G-protein recruitment assays on nanodisc-encapsulated receptors, we show that all three copolymers maintain high-affinity extracellular ligand binding but differ markedly in their ability to preserve intracellular signalling. Despite lower receptor extraction efficiency, only sulfo-DIBMA support mini-Gs engagement at the CGRP receptor and enable G-protein-dependent allosteric modulation at the PTH1 receptor, including conserved ligand affinity and prolonged residence time. These data reveal that polymer charge and backbone chemistry, rather than extraction yield, determine whether native-like nanodiscs retain the conformational landscape required for active-state signalling. Controlling non-specific ligand binding to the copolymer is a key requirement for a successful assay. Our findings identify sulfo-DIBMALP as a particularly superior environment for preserving native signalling behaviour in class B1 GPCRs, highlighting copolymer chemistry as an important determinant in detergent-free membrane protein studies. HIGHLIGHTSO_LISulfo-DIBMA encapsulated nanodiscs preserve active-state conformation of human calcitonin gene-related peptide receptor and parathyroid hormone 1 receptor. C_LIO_LIAll three copolymers (SMA 2000, DIBMA-12 and sulfo-DIBMA) preserve extracellular ligand binding but only sulfo-DIBMA preserves intracellular functional competence, including mini-Gs recruitment and G-protein-dependent allosteric modulation. C_LIO_LICopolymer chemistry, particularly the electroneutral, aliphatic nature of sulfo-DIBMA, may influence the preservation of signalling-competent states in two class B1 GPCRs by minimising charge-driven perturbations during solubilisation. C_LIO_LISulfo-DIBMALP provides a novel platform for studying dynamic membrane proteins with potential to provide mechanistic insights and facilitate drug discovery programmes in the future. C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=103 SRC="FIGDIR/small/724797v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@12db163org.highwire.dtl.DTLVardef@d8efb3org.highwire.dtl.DTLVardef@610dbaorg.highwire.dtl.DTLVardef@1cc3ce4_HPS_FORMAT_FIGEXP M_FIG C_FIG

Activity-based probes (ABPs) are widely used to profile serine protease activity - enzymes central to diverse physiological and pathological processes - but most rely on covalent modification of the conserved catalytic serine residue, often resulting in poor selectivity across related proteases. Here, we introduce covalent macrocyclic activity-based probes (cmABPs) that selectively target non-catalytic residues within serine protease active sites. By combining phage display with systematic electrophile scanning, we identify macrocyclic scaffolds that position sulfur(VI) fluoride (SuFEx) electrophiles to covalently engage alternative nucleophiles such as lysine and tyrosine. Applied to plasma kallikrein, this approach yielded a macrocyclic scaffold that was converted into covalent probes via fluorosulfate scanning. Remarkably, small changes in electrophile structure produced large, tuneable differences in covalent kinetics, with benzenesulfonyl fluoride derivative 23 achieving rapid and complete protein modification. Biochemical and mass spectrometry analyses confirmed selective modification of an active-site lysine by 23, along with robust performance in complex biological samples. Extension to urokinase plasminogen activator further demonstrates the generality of this strategy. More broadly, this work establishes electrophile scanning within macrocyclic scaffolds as a general approach for tuning covalent reactivity and provides a blueprint for designing selective probes that move beyond catalytic-residue targeting.

Aberrant HGF-c-MET signaling is a major driver of hepatocellular carcinoma (HCC) progression and a clinically validated therapeutic axis, but current inhibitors predominantly target the intracellular kinase domain and remain vulnerable due to limited selectivity and resistance development. We therefore pursued an upstream strategy based on small molecules that target the extracellular HGF-c-MET interaction interface. We combined large-scale virtual screening of more than one million compounds from the ChemDiv and Enamine libraries with molecular dynamics (MD) simulations, steered MD, MM/GBSA profiling, and iterative lead optimization to identify candidate c-MET inhibitors targeting its extracellular (EC) domain. In HGF-stimulated HuH7 cells, selected compounds suppressed c-MET autophosphorylation, reduced cell viability, and inhibited long-term colony formation. Surface plasmon resonance (SPR) further confirmed direct binding of L083-1287 and 8008-3424 to the recombinant c-MET ectodomain. Mechanistic analyses identified previously unrecognized hotspot residues on the c-MET EC domain and a novel inhibitory network spanning multiple c-MET ectodomain interfaces. L083-0077 displayed the most consistent interaction pattern within this framework, including stabilization of key hotspot residues and preserved binding under acidic conditions relevant to the tumor microenvironment. Zebrafish xenograft assays with selected early hit compounds revealed compound-dependent developmental liabilities supporting the use of this model as an early in vivo prioritization step during lead optimization. These findings establish EC interface-directed c-MET inhibition as a promising therapeutic strategy in HCC and provide a mechanism-guided platform for the development of selective, upstream c-MET inhibitors with the potential to complement or overcome limitations of kinase-directed therapies.

Positive allosteric modulators (PAMs) of the M4 muscarinic acetylcholine receptor (mAChR) represent a promising therapeutic strategy for treating cognitive deficits and neuropsychiatric disorders. While first-generation M4 mAChR PAMs, like LY2033298, demonstrated proof-of-concept, second-generation compounds, such as MK-97, exhibit substantially improved potency and reduced species variability. Here we report the cryo-EM structure of the M4 mAChR bound to the endogenous agonist, acetylcholine, and MK-97 at 2.7 [A] resolution, revealing the molecular basis for improved M4 mAChR PAM activity. MK-97 adopts a distinctive boomerang-shaped conformation within the extracellular-facing allosteric binding site, with a central pyridine vertex, a lower cyclopentylmethylpyrazole arm extending toward the floor of the orthosteric site, and an upper isoindolinone arm projecting toward extracellular loop 2 (ECL2). This extended binding mode establishes a distributed interaction network across transmembrane helices TM2, TM3, TM5, TM6, and TM7, with key contacts including a hydrogen bond with Y922.64 and a {pi}-{pi} stacking interaction with W4357.35. Integration of structural data, molecular dynamics simulations, and mutagenesis validation reveals that the high affinity of MK-97 derives from optimized engagement across all three binding regions rather than dependence on any single critical contact. Insights from comprehensive structure-activity relationship (SAR) studies provide a molecular framework for the rational design of next-generation M4 mAChR PAMs with improved pharmacological properties. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=70 SRC="FIGDIR/small/723386v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@1ab9c78org.highwire.dtl.DTLVardef@1adb532org.highwire.dtl.DTLVardef@152f9f7org.highwire.dtl.DTLVardef@990768_HPS_FORMAT_FIGEXP M_FIG C_FIG

Modern protein design methods based on deep learning allow generation of customized protein scaffolds with diverse geometries and functionalities. Here, we capitalize on these recent advances to develop hyper-thermostable de novo CO2 reductases featuring a cobalt porphyrin IX cofactor (CoPPIX). CoPPIX containing enzymes were assembled in vivo through media supplementation with cobalt salts and assessed for photocatalytic CO2 reductase activity. We identified two cysteine-ligated designs that exhibit high activity (>1000 turnovers at rates of up to 25 min-1) while suppressing competing hydrogen evolution pathways. A 2.1 [A] crystal structure shows close agreement to the design model with the Co-Cys bond programmed as intended. This study showcases the power of computational protein design in developing artificial enzymes to activate challenging molecules such as CO2.

Crystalline inclusion proteins CipA and CipB from Photorhabdus luminescens serve as versatile scaffolds for clustering genetically fused heterologous enzymes into crystalline inclusion bodies. Although engineered Cip crystals are known to function as solid biocatalysts for improving metabolite production in bacterial cells, the phase separation behavior of Cip proteins in non-bacterial cellular environments, as well as their biochemical attributes in a soluble, non-crystalline state, remain poorly understood. This study demonstrates that CipA and CipB efficiently undergo crystallization in the cytosol of human embryonic kidney cells both at normal and hypothermic cell culture conditions. Within 72 hours post-transfection, CipA and CipB become the most abundant proteins in transfected cells and produce distinctive cytosolic crystals often exceeding 10 m at least in one of the dimensions. Co-expression of CipA and CipB drives spontaneous demixing into two distinct crystal populations, and the orthogonality is maintained even when an unrelated third protein crystallizes in the same cytosol, permitting three crystal types to coexist simultaneously. Intracellular crystals are readily isolable from cells, and once purified, these crystals are stable under physiological pH conditions. However, CipA and CipB show notable differences in their crystal dissolution kinetics and protein oligomerization states when solubilized under acidic or alkaline conditions. These findings suggest that CipA/CipB forms a robust orthogonal self-assembly pair and establish CipA/CipB crystals as an efficient platform for producing biochemically programmable intracellular crystals. These properties should extend the Cip-based scaffolding approach to mammalian cell systems for synthetic biology applications.