Biochemistry

Acetyl-coenzyme A (CoA) is a central metabolic intermediate that links carbon and energy metabolism across all domains of life. The conversion of acetate and acetyl-CoA is carried out by three enzyme pathways: acetate kinase/phosphotransacetylase, ADP-forming acetyl-CoA synthetase, and AMP-forming acetyl-CoA synthetase (Acs). Acs enzymes serve critical physiological roles across diverse organisms generally by catalyzing a reversible two-step reaction forming acetyl-CoA and AMP from acetate and ATP. Isolated from the wastewater reclamation facility in Norman, Oklahoma, Syntrophus aciditrophicus strain SB (Sa) relies on an AMP-forming acetyl-CoA synthetase (SaAcs1) that favors synthesizing acetate and ATP from acetyl-CoA and AMP, in contrast to all previously characterized Acs enzymes. The origin of this preference and the structural determinants of both the thioester-forming step and catalytic directionality remain poorly understood. Here, we report a 2.2 [A] crystal structure of full-length SaAcs1 in the adenylation conformation with acetyl-AMP bound in the active site. Structural comparison to the extensively characterized Acs enzymes from Salmonella enterica (SeAcs) and Cryptococcus neoformans (CnAcs) revealed a displaced CoA-binding loop in SaAcs1. Enzymatic assays confirmed that SaAcs1 preferentially catalyzes the ATP-forming reaction. Site-directed mutagenesis demonstrated that reversion of two residues, G196 and T197, at the beginning of the CoA-binding loop to the consensus sequence repositions the loop and shifts catalytic preference toward the AMP-forming direction. Together, these results establish the CoA-binding loop and G196 and T197 as the primary structural determinants of directional preference in SaAcs1.

Antimicrobial resistant infections present a growing threat to public health and were associated with or directly caused 6 million deaths globally in 2021. This huge death toll highlights the need for novel strategies to address AMR infections. Interfering with the regulation of resistance mechanisms could provide an alternative approach to treat drug-resistant infections. PhoQ, a sensor histidine kinase ubiquitous amongst gram-negative bacteria, regulates several virulence factors, as well as resistance to outer membrane-targeting antibiotics, making it an attractive target for adjuvant therapy development. However, the identification of potent small molecule inhibitors is limited by the assays available for in vitro assessment of binding and activity inhibition in PhoQ. Thus, we sought to investigate the use of a fluorescence-based assay to evaluate enzymatic activity, as well as a thermal shift assay to assess inhibitor-protein binding in PhoQ. Together, these newly implemented protocols are valuable contributors to the toolbox of methods available for the development of PhoQ-targeted inhibitors to block this major contributor to antimicrobial resistance.

AMP deaminase (AMPD) plays an integral role in fructose metabolism via its regulation by ATP, GTP and phosphate (Pi). The fructose catabolic pathway consumes ATP, producing ADP, which is further metabolized to AMP, triggering a cascade of reactions initiated by AMPD. This degradative pathway results in the final product uric acid, which is associated with metabolic acidosis, mitochondrial dysfunction, and gout. Understanding the regulation of the human liver AMPD isozyme (hAMPD2-2) under physiological conditions and under fructose consumption conditions will enable the design of targeted therapeutics to block the accumulation of uric acid. We report the first successful expression and purification from Escherichia coli of both the full-length and catalytic domains of hAMPD2-2. Steady-state kinetics confirmed allosteric activation by ATP of both the full-length and catalytic domains of hAMPD2-2 at physiological ATP concentrations (2-5 mM), suggesting that the allosteric ATP-binding site is located in the catalytic domain. Competitive inhibition by GTP of the ATP-activated enzyme, with Ki values of 74 and 101 M for the full-length and catalytic domains, respectively, was also consistent with this regulatory model. Pi, previously described in yeast AMPD as a competitive inhibitor, was shown to play a more nuanced role, that of enhancing inhibition of hAMPD2-2 when the enzyme is complexed to GTP, via competition at the ATP allosteric site. Pi binding thus further inhibits the pathway under normal physiological conditions, limiting production of cellular uric acid unless and until Pi and GTP levels are low.

Post-translational modifications usually occur under enzymatic control as a mechanism for regulation of protein activity or localization, but can also occur spontaneously during protein aging and degradation. In contrast, there are few examples of spontaneous post-translational modifications with a significant role in the stability or biochemical function of a bacterial protein. Here we show that a spontaneous post-translational modification is structurally encoded and functionally important in a bacterial solute-binding protein. We show that the solute-binding protein SAR11_0655 from the abundant SAR11 marine bacterium Pelagibacter ubiqueversans contains an unusually stable succinimide residue important for its function in high-affinity binding of the amino acid derivative 5-oxoproline. Spontaneous cyclization of Asn269 to form a succinimide residue was inferred from high-resolution X-ray crystallography and confirmed by liquid chromatography-tandem mass spectrometry. Loss of the succinimide modification via the substitution N269A resulted in a >20 {degrees}C decrease in thermostability and a 15 to 50-fold decrease in binding affinity for 5-oxoproline. Analysis of sequence conservation indicated that both Asn269 and Asp269 are common among SAR11_0655 homologs, and the N269D variant of SAR11_0655 also showed partial formation of succinimide. These results reveal a previously unrecognized mechanism by which proteins exploit spontaneous post-translational chemistry as a design feature to enhance stability and function, and provides a potential basis for identification and design of structurally encoded succinimide residues for engineering of protein thermostability.

Natural product (NPs) and their derivatives are a major source of small-molecule drugs, and the building blocks of these NPs are often amino acids. These include both proteinogenic and nonproteinogenic amino acids (NPAAs), the latter of which expand the structural diversity of NPs. Homologation, or the addition of a methylene group to the amino acid side chain, is one modification that generates NPAAs. If the natural homologation pathway can be characterized and engineered, it could be used to diversify NPs. In this study, we investigated the terminal enzyme of this pathway, HphB, to determine its substrate scope. HphB was tested with various substrates that differed in backbone and/or side chain structures relative to its natural substrate. The results showed that HphB exhibits high promiscuity toward substrates with different side chains while maintaining strict specificity for the substrate backbone. Comparative analysis with two homologous enzymes from primary metabolic pathways revealed that HphB displays markedly higher substrate promiscuity. Bioinformatics analysis and structural modeling suggest that this promiscuity arises from the absence of a "lid" over the active site, resulting in increased solvent exposure of the substrate side chain. This study highlights the unique substrate flexibility of HphB and is a step toward engineering the homologation pathway to generate amino acid derivatives.

Phycobiliproteins form oligomeric assemblies essential for photosynthetic light harvesting. Here, we engineered phycocyanin (TeCPC) and allophycocyanin (TeAPC) from Thermosynechococcus elongatus to stabilize defined trimers by inhibiting hexamer formation. Structure-guided substitutions at conserved glycine residues (TeCPC G29R, TeAPC G21R) introduce steric hindrance at the hexamer interface. Recombinant expression in Escherichia coli produced holoproteins with native-like chromophorylation. Biophysical and structural analyses confirmed homogeneous trimer formation and absence of higher-order assemblies. Thermal measurements indicated cooperative unfolding, supporting structural uniformity. These engineered trimers provide robust models for studying energy transfer in phycobiliproteins.

The biosynthetic machineries of ribosomally synthesized and post-translationally modified peptides (RiPPs) are often substrate tolerant. A remarkable example is the class II lanthipeptide synthetase ProcM, which naturally functions as a generalist enzyme that has not evolved to use a specific substrate during its evolutionary history. Although ProcM has been studied extensively, the sequence features associated with productive modification remain underexplored. In this study, we use the ultrahigh-throughput mRNA display technique to map the sequence compatibility of ProcM across a focused library. This approach expands the landscape of ProcM reactivity beyond native substrates and individually characterized variants. Machine learning (ML) is used as a tool to demonstrate that the selected dataset contains learnable signatures and classification architectures revealed a balanced accuracy of 0.73. This performance contrasts sharply with the near-perfect accuracy of specialized enzyme models as the sequence-fitness landscape of the generalist enzymes are characterized by class imbalance and limited by intrinsic dataset features. Our results provide a high-throughput view of ProcM reactivity and highlight differences with previous high-throughput studies on substrate selectivity of RiPP modification enzymes. Future studies will need to assess whether these differences are common when comparing generalist with specialist enzymes.

2-Deoxyribose-5-Phosphate Aldolase (DERA) is a key enzyme in the pentose phosphate pathway. Due to its C-C bond formation and stereoselective capabilities, DERA has been widely used for biocatalytic applications including the synthesis of chiral intermediates for antiviral and anticancer drugs. While protein engineering has expanded its substrate pool, improved yield, and enhanced stereoselectivity, the molecular basis of stereoselectivity remains unclear. Here, we determined the crystal structures of wildtype DERA from Geobacillus sp. and two of its variants with opposite stereoselectivity. Using a combination of structural biology, biochemistry, organic synthesis and molecular dynamic simulations, we show that the catalytic Lysine adopts two conformations and the Lysine conformation is a key determinant of DERA stereoselectivity. We also identified a mechanism of regulating stereoselectivity via a key amino acid. Using DERA from E. coli, we show that these findings are most likely conserved among bacteria.

Sliding clamp proteins play central roles in DNA metabolism, including replication and repair. The ring-shaped E. coli beta clamp accommodates double-stranded DNA and serves as a platform for proteins involved in multiple DNA transactions. The inner pore of the beta clamp harbors a series of positively charged and polar residues that can bind to the negatively charged backbone of the DNA. These residues are arrayed so that they do not align with the charged phosphates of the DNA backbone. It is hypothesized that this arrangement of these residues provides for the movement of the clamp on DNA as it alternates which residues are bound to the DNA backbone. In this work, we mutated specific charged and polar residues that project into the inner pore of the beta clamp. The beta clamp variants are dimers and have similar thermal stability and in general a similar ability to complement a temperature sensitive strain for growth. One exception was beta-Q149A, which appeared as higher-order species on a native gel although its hydrogen-deuterium exchange pattern measured by mass spectrometry was overall similar to WT beta. These variants all had decreased binding to DNA after loading. Optical tweezers experiments were used to monitor loading on single DNA molecules and measure the rate of beta clamp sliding on DNA. Consistent with the hypothesized role of positively charged residues in the beta inner pore, mutation of one residue resulted in a faster rate of sliding on DNA.

Conversion of cysteine residues to 5-thiooxazole moieties by multinuclear nonheme iron-dependent oxidative enzymes (MNIOs) is a prevalent modification in ribosomally synthesized, post-translationally modified peptide (RiPP) natural products. However, this post-translational modification (PTM) is difficult to distinguish from MNIO-produced oxazolone-coupled thioamides, such as those present in the RiPP methanobactin. The RiPP virulence factor oxazolin contains six copper-binding heterocycles installed by an MNIO. Here, we reassign these PTMs, originally described as oxazolones/thioamides, as 5-thiooxazoles on the basis of detailed comparative chemical and structural characterization of oxazolin and methanobactin. These data establish a benchmark for differentiating these two PTMs in newly discovered RiPPs. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=86 SRC="FIGDIR/small/730506v1_ufig1.gif" ALT="Figure 1"> View larger version (12K): org.highwire.dtl.DTLVardef@d30435org.highwire.dtl.DTLVardef@1b933fforg.highwire.dtl.DTLVardef@4b997org.highwire.dtl.DTLVardef@349ee1_HPS_FORMAT_FIGEXP M_FIG C_FIG

BRCA1/BARD1 is a chromatin-associated E3 ubiquitin ligase that ubiquitylates histone H2A to coordinate DNA damage repair, transcriptional repression, and genome stability. In Caenorhabditis elegans (C. elegans), the orthologous BRC-1/BRD-1 complex performs analogous functions but exhibits structural variation, most notably through an additional 11-residue loop in BRD-1 that is absent from human BARD1. Prior experiments indicate this worm-specific insertion promotes nucleosome engagement and may alter the preferred lysine target for ubiquitylation. Here, we provide a cross-species comparison by integrating computational and experimental investigation to clarify how a discrete structural variation can tune BRCA1-family ligase behavior and, consequently, chromatin regulation. In vitro ubiquitylation assays and mass spectrometry reveal BRC-1/BRD-1 ubiquitylate the C-terminal tail of histone H2A with less specificity than the human homologs. All-atom molecular dynamics simulations of both the C. elegans BRC-1/BRD-1-LET-70-Ubiquitin assembly and the human BRCA1/BARD1-UbcH5c- Ubiquitin complex in the presence of the nucleosome core particle uncover that the BRD-1 loop makes transient contacts with nucleosomal DNA and histone tails, thereby modulating the positioning and conformational flexibility of the bound E2 (ubiquitin-conjugating enzyme). Together, our results suggest that the BRD-1 loop alters the E3-E2 geometry, thereby altering ubiquitylation-site specificity.

Quorum sensing in Gram-positive bacteria commonly relies on posttranslationally modified peptide pheromones. In Bacillus subtilis, the prenyltransferase ComQ catalyzes tryptophan prenylation of the quorum-sensing peptide ComX, but the structural basis of this unique peptide modification has remained unclear. Here we identified a previously uncharacterized ComQ homolog, StheQ, and its cognate peptide substrate, StheX, from Sphaerobacter thermophilus and investigated their structural and functional relationship. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis demonstrated that StheQ catalyzes prenylation of the tryptophan residue located second from the C-terminus of StheX. Crystal structures of apo StheQ and its complexes with a farnesyl pyrophosphate analog revealed that StheQ adopts the all--helical fold of the trans-isoprenyl diphosphate synthase (IPPS) superfamily while possessing an active-site architecture adapted for peptide-based indole prenylation. The structures identified a single Mg2+-binding site associated with the first aspartic acid-rich motif and showed no evidence for metal coordination at the pseudo-second aspartic acid-rich motif. Site-directed mutagenesis, complex formation assays, and docking analyses identified a peptide-binding pocket adjacent to the active site and suggested that N215 contributes to productive positioning of the acceptor tryptophan. These findings establish the structural basis for peptide prenylation by a ComQ-family enzyme, providing insight into the evolution of peptide-based indole prenylation within the IPPS superfamily, and support the view that ComQ-family enzymes constitute a distinct functional branch specialized for peptide modification.

Cnidarians possess large number of opsins in their genomes for their various photoreceptive functions. In particular, they uniquely possess Gs-coupled opsins that induce intracellular cAMP accumulation in a light-dependent manner. These Gs-coupled opsins, cnidopsins, are powerful optogenetic tools manipulating cAMP-dependent cellular responses. In this study, we characterized a cnidopsin, named as AtCnidop3a, from the coral Acropora tenuis as a Gs-coupled and UV-sensitive bistable pigment. This cnidopsin showed a large spectral shift upon activation from absorption maxima from 395 nm to 560 nm, and the resting and activated states are interconvertible by illumination with UV (or violet) and orange light. The activated state efficiently activated Gs proteins and elevated intracellular cAMP levels in mammalian cultured cells. To engineer the opsin mutant that can be turned on and off upon long wavelength light illumination by utilizing the large spectral separation, negatively charged amino acids were introduced near the retinal Schiff base region. Among tested opsin mutants, the Y1133.28E mutant is capable of being activated by green light unlike the wild-type while retaining the property of being inactivated by orange light like the wild-type, indicating successful conversion of the opsin to a visible light sensitive bistable pigment. The visible light-induced cAMP regulation of the Y1133.28E mutant was enhanced by an additional L942.61G substitution. Our characterization and engineering of the cnidopsin revealed functional diversity of cnidarian opsins and its potential utility as optogenetic tools regulating Gs-dependent physiological responses.

Inter-subunit communication and allosteric regulation are central to the function of oligomeric enzymes, yet these features remain difficult to characterize. Conventional kinetic and structural methods typically yield ensemble averages or static snapshots, thus making it difficult to uncover the dynamic cross-subunit cooperation obligatory for multi-site catalysis by oligomeric enzymes. Here, we investigate Salmonella FraB--a homodimeric deglycase and a potential drug target--to showcase the value of an integrated approach combining native mass spectrometry (nMS), surface-induced dissociation (SID), and kinetic studies to gain insights into catalytic intermediates and inter-subunit communication. By resolving substrate-, product-, and mixed-occupancy species, nMS revealed that both inter-subunit active sites in FraB bind substrate even though only one catalytic center generates the product at any given time. To characterize each active site independently, we designed heterodimers with a mutation that changes the general base or acid in only one active site. Kinetic studies with these mutants indicate that although the two active sites are likely coupled, they do not concomitantly perform cleavage. Consistent with the conformational asymmetry observed in apo-FraB crystal structures, our findings establish a half-site reactivity mechanism in which post-binding conformational changes across the dimer interface restrict substrate cleavage to one active site even though both protomers are able to bind substrate. Importantly, this nMS-based workflow offers a broadly applicable framework for resolving the catalytic states and inter-site communication of oligomeric enzymes that are otherwise difficult to uncover by conventional structural methods.

Bacterial sortases are widely used in sortase-mediated ligation (SML) experiments for various protein engineering applications. The power of these enzymes to bind and cleave a specific recognition motif, followed by ligation to another substrate using a ping-pong reaction mechanism has numerous applications in vaccine and antibody/nanobody drug conjugate development, as a diagnostic and therapeutic tool, in creating novel insulin derivatives, etc. The most widely used sortase for SML is the class A sortase (SrtA) from Staphylococcus aureus (saSrtA), and its engineered derivatives. Despite its utility, saSrtA and other endogenous sortases are relatively inefficient enzymes and use can be limited by the need for specific recognition of the Cell Wall Sorting Signal (CWSS), sequence Leu-Pro-X-Thr-Gly, where X=any amino acid. Therefore, there is a need to continue to identify new tools for SML and to develop screening assays towards these endeavors. Here, we present optimization procedures for a FRET-based assay utilizing the GFP derivatives mTurquoise2 and SYFP2 to directly monitor formation of ligation products generated via SML. Similar to related assays, our recombinant substrates can be easily manipulated to screen either the substrate recognition motif, second substrate nucleophile, and/or sortase variants themselves. We believe continued optimization of this assay for a variety of high throughput uses in sortase screening strategies is possible, providing a proof-of-concept approach for continued SML reagent development.

T cells are important components of the adaptive immune system and develop through a selection process regulated by signaling through the T-cell receptor (TCR). Thymocyte-Expressed Molecule Expressed in Selection (THEMIS) is a TCR-proximal protein that modulates the activity of Shp1 phosphatase to influence TCR signaling during development. THEMIS has been shown to both activate and inhibit Shp1, but the molecular mechanisms of these functions are poorly understood. THEMIS contains two rare Cysteine All-Beta In THEMIS (CABIT) domains, the N-terminal of which interacts with Shp1 and is likely responsible for modulation of its phosphatase activity. Herein, we report the first crystal structure of the THEMIS CABIT1 domain. While a portion of the CABIT1 domain is poorly resolved, it appears to share the same overall fold observed in our recent CABIT2 crystal structure and AlphaFold predictions. We show that phosphorylation of the CABIT1 domain by LCK is required for association with SHP1 and that phosphorylated CABIT1 can protect Shp1 from oxidation and inhibition by reactive oxygen species (ROS), which may serve as a mechanism by which THEMIS enhances Shp1 activity.

The multinuclear nonheme iron-dependent oxidative enzyme (MNIO) family employs a multi-iron cofactor to catalyze a range of post-translational modifications (PTMs) in the biosynthesis of ribosomally synthesized, post-translationally modified peptide (RiPP) natural products. While significant progress has been made toward understanding the range of chemical transformations performed by MNIOs, the nature of the iron cofactor has only been investigated in one instance. Here, we examine the MNIO involved in oxazolin biosynthesis to gain further insight into the metallocofactors employed by this impressive family of enzymes. Oxazolin, a RiPP virulence factor from nontypeable Haemophilus influenzae, contains six copper-binding 5-thiooxazole groups installed by the MNIO HvfB. Weak interactions between HvfB and its required partner protein, HvfC, motivated genetic fusion of the two proteins, which yielded an effective mimic of the protein complex with high enzymatic activity. While HvfB binds up to three iron ions, concerted EPR, ENDOR, and Mossbauer spectroscopic characterization of the active protein reveals that accumulation of a mixed-valent diiron(II/III) cluster correlates with 5-thiooxazole product formation. This oxidation state is attained only in the presence of HvfC, revealing a new role for the partner protein in modulating the iron cofactor. Site-directed mutagenesis of metal-coordinating residues was used to probe the function of the third iron-binding site. This work clarifies the nature of the active iron cofactor for oxazolin maturation, providing a second example of a mixed-valent diiron oxidase in RiPP biosynthesis. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/739211v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@bf1da7org.highwire.dtl.DTLVardef@133f196org.highwire.dtl.DTLVardef@139e8eaorg.highwire.dtl.DTLVardef@ea5f61_HPS_FORMAT_FIGEXP M_FIG C_FIG

-Synuclein (S), a protein that plays a central role in Parkinsons disease and related synucleinopathies, is an intrinsically disordered protein (IDP) whose functional interactions and aggregation behavior can be strongly influenced by post-translational modifications (PTMs). Phosphorylation, acetylation, and other PTMs regulate Ss interactions with lipid membranes and binding partners, whereas their dysregulation is associated with aggregation and neuronal toxicity. Despite significant progress through chemical and semi-synthetic approaches, investigating the combinatorial effects of PTMs has remained challenging due to the lack of accessible, site-specific methods. Here, we present an integrated strategy combining genetic code expansion, enzymatic modification, and intein-mediated click chemistry to generate S variants bearing multiple defined PTMs and a C-terminal fluorescent label. The resulting constructs enable direct evaluation of how individual and combined PTMs influence S structure, lipid binding, and cellular internalization. Our approach expands the molecular toolkit for dissecting PTM crosstalk in S and other aggregation-prone IDPs, advancing mechanistic understanding and supporting the development of therapeutic strategies for neurodegenerative disease.

Oxalyl-coenzyme A (oxalyl-CoA) is a key intermediate in oxalate metabolism in plants, fungi and oxalate-degrading bacteria, but its limited availability has restricted biochemical investigations of oxalyl-CoA-dependent enzymes. Here, we describe a practical semisynthetic procedure for the preparation of oxalyl-CoA based on rapid oxalyl transfer from S-oxalyl p-thiocresol to coenzyme A. The reaction was monitored directly by 1H NMR spectroscopy, allowing optimisation of pD and reaction conditions. Following removal of thiocresol and purification by reversed-phase HPLC, oxalyl-CoA was obtained in 39% yield as determined by quantitative 1H NMR. The product was characterised by high-resolution electrospray mass spectrometry and comprehensive 1H, 13C and 31P NMR spectroscopy, confirming its structure unequivocally. During the study, the limited stability of oxalyl-CoA in aqueous solution was documented, leading to recommendations for its purification and storage. The semisynthetic protocol provides a convenient source of analytically pure oxalyl-CoA suitable for biochemical assays and supplies reference spectroscopic data for its unambiguous identification. The biological utility of the semisynthetic oxalyl-CoA was demonstrated by its application as an acyl donor substrate in assays of PnBAHD15, enabling quantitative kinetic characterisation of the enzyme and illustrating its suitability for biochemical studies of oxalyl-CoA-dependent enzymes.

Monobodies are engineered binding proteins that recognize extended protein surfaces and offer advantages over small-molecule inhibitors for targeting challenging KRAS oncoproteins. Monobody 12D4 exhibits high affinity and selectivity for the oncogenic KRAS(G12D) mutant, but the molecular determinants governing its recognition and the basis for its mutant selectivity remain poorly understood. Here, we combined molecular dynamics simulations and energy calculations to characterize the interactions between monobody 12D4 and WT KRAS as well as four clinically relevant oncogenic variants (G12C, G12D, G12V, and G12R) in both GTP- and GDP-bound states. Our simulations revealed that 12D4 recognition depends on a conserved hydrophobic interaction network centered on the monobody FG loop (residues L77, F78, and W79). This network forms stable contacts with KARS Switch II and 3-helix. The energy calculations also showed that residue K75 of 12D4 formed a mutation-specific electrostatic interaction with KRAS G12D. This interaction contributed significantly to the affinity of 12D4 toward this mutant, whereas this interaction was absent in other variants. No monobody currently exists for targeting KRAS G12R in either nucleotide state, and no monobody selectively targets KRAS G12C and G12V in the GDP-bound inactive state. To address these, we performed computational redesign at residues 75. We identified mutations (K75Q, K75Y, and K75M) that enhanced predicted binding to G12C, G12R, and G12V variants through reorganization of interfacial contacts. Our work establishes a structural framework for understanding KRAS-monobody recognition and provides a rational foundation for engineering variant-selective monobodies with improved affinity toward previously untargetable KRAS mutants.