Biology subjects

Kuriyan, J.

Publications and source records attributed to Kuriyan, J..

At least 19 recordsLinked to original sources

bioRxiv2026

A rational design strategy and validation for protease-resistant fusion-inhibitor antiviral peptides

Peptide-based fusion inhibitors are promising pharmaceuticals in the fight against enveloped viruses relying on membrane fusion for host infection. However, peptide therapeutic applications have long been hindered by their poor stability in vivo. Here, we discovered that peptide inhibitors with the wildtype sequence of the heptad repeat 2 (HR2) domain of the SARS-CoV-2 spike protein are efficiently cleaved by Transmembrane Protease, Serine 2 (TMPRSS2), a key protease involved in the SARS-CoV-2 virus-cell fusion pathway. We then identified the corresponding cleavage sites and designed three protease-resistant peptides using ranking based on deep mutational scanning and natural occurrence. The three candidates all exhibit inhibitory activity in a cell-cell fusion assay. A high-resolution cryo-EM structure of the top candidate, HR2-NHN, bound to its HR1 target reveals the molecular basis for its potent activity. The top candidate of the cell-based screening assay significantly improved efficacy relative to the wildtype peptide when administered 12 h before infection in both an authentic virus-cell infection assay and a mouse assay. More broadly, our results suggest that the design strategies for protease-resistant peptides could be applied to a broad spectrum of other enveloped viruses and pave the way for the development of safe, prophylactic antivirals that can be administered before exposure.

microbiology↗

bioRxiv2026

Redesign of energetically frustrated regions rescues function in defective T4 clamp loaders

DNA polymerase clamp loaders are AAA+ ATPases that load sliding clamps on DNA for high- speed replication. Using a platform for high-throughput mutagenesis of replication proteins in T4 bacteriophage, we carried out saturation mutagenesis of the AAA+ ATPase module of the T4 clamp loader bearing a mutation, Gln 118{lozenge}Asn (Q118N), that reduces fitness. We identified residues for which different mutations improve the fitness of the Q118N variant but are neutral in the wild-type background. These conditionally neutral "rescue hotspots" overlap with those identified earlier in another defective variant (D110C). These rescue hotspots localize to regions where the sequence is not optimal for the structure, as determined by energetic frustration analysis. We designed new sequences for three of these regions, using the protein-design algorithm ProteinMPNN. In two helical regions, several designed sequences increased the fitness of both wild-type and mutant proteins, likely due to enhanced stability. An inter-domain hinge in AAA+ module changes conformation during activation, and designs for the hinge lead to loss of fitness in the wild-type background. However, when using the active conformation as the template, designs for the hinge increase the fitness of defective variants. In contrast designs templated on the inactive conformation led to loss of fitness, suggesting that a proper conformational balance is crucial. Thus, adaptive capacity in the clamp loader resides in a network of conditionally neutral sites that enable functional tuning through shifts in stability and conformational equilibria.

biochemistry↗

bioRxiv2026

LAT condensation gates PLCγ1 activation via bimodal LAT phosphorylation

T cells can respond to even a single molecular binding event of antigen to a TCR. A key step in the TCR signaling pathway that definitively exhibits this single molecule response is the initiation of calcium influx by activation of PLC{gamma}1 in the LAT protein condensate. Here, we describe detailed kinetic measurements examining how protein condensation of LAT regulates activation of PLC{gamma}1 using a reconstituted membrane system. The results reveal that membrane recruitment of PLC{gamma}1 is tightly controlled by the LAT phosphorylation state, with no measurable independent recruitment to PIP2 or PIP3 lipids via the PLC{gamma}1 PH domains. We further observe PLC{gamma}1 is rapidly activated by membrane-associated kinase upon recruitment, irrespective of the LAT condensation state. These studies also revealed a crosstalk mechanism in which the TEC family kinases responsible for PLC{gamma}1 activation also phosphorylate LAT. This interaction establishes a positive feedback loop in LAT phosphorylation, mediated through LAT condensation, which drives a bimodal LAT phosphorylation response to TCR activation. Kinetic modeling reveals how this LAT phosphorylation response can cooperatively gate PLC{gamma}1 activation from a single TCR. These results suggest the LAT condensate facilitates both signal amplification and noise suppression in PLC{gamma}1 activation through a bimodal switch affecting LAT phosphorylation. Significance StatementT cells are sensitive sensors capable of detecting and responding to trace amounts of foreign antigen. Understanding how they achieve such sensitivity while maintaining accurate antigen discrimination remains a key challenge. Here, through detailed kinetic measurements of PLC{gamma}1 activation, we identify a cross reactivity in which kinases responsible for PLC{gamma}1 phosphorylation also phosphorylate LAT. This creates a bimodal switch controlling LAT phosphorylation levels, which gates PLC{gamma}1 activation from single TCR signals. We suggest this mechanism plays a key role in the signal amplification and noise suppression required for T cells to detect single antigen molecules.

biophysics↗

bioRxiv2026

The Central Coupler of the AAA+ ATPase ClpXP Controls Intersubunit Communication and Couples the Conversion of Chemical Energy into the Generation of Force

ClpX is a clockwise hexameric helical arrangement that hydrolyzes ATP to unfold proteins and translocate them into the proteolytic chamber. We investigate the central coupler, a three -helix module conserved among AAA+ ATPases, which is proposed to enable intersubunit communication and mechanochemical coupling. Although fundamental in AAA+ ATPases, the molecular mechanism underlying these processes remains elusive in ClpX. By combining single-molecule optical tweezers, biochemical assays and single particle cryo-EM we demonstrate that the central coupler, of the second-highest subunit of the ClpX clockwise helical arrangement, positions its Arginine-finger, sensor-I and sensor-II to contact the ATP of the anticlockwise neighbor subunit to trigger its hydrolysis. Then, the central coupler of the highest subunit rapidly couples the ATP hydrolysis with the downward motion of its substrate-translocating loop, enabling fast force generation to swiftly unfold protein substrates while maximizing the thermodynamic efficiency of the motor.

biophysics↗

bioRxiv2025

BTK Autoinhibition Analyzed by High-Throughput SH2 Domain Swaps

BTK, a Tec-family tyrosine kinase, resembles the Src and Abl kinases in that an SH2-SH3 module regulates the activity of the kinase domain, principally through an inhibitory interaction between the SH3 and kinase domains. In Src kinases, phosphorylation of a C-terminal tail latches the SH2 domain onto the kinase domain, positioning the SH3 domain in an inhibitory conformation; in Abl, interaction between the kinase domain and a myristoyl group on the N- terminal segment provides the same latching function. The structure of autoinhibited BTK resembles that of the Src and Abl kinases, but BTK lacks an SH2-kinase latch. To assess autoinhibition in BTK, we generated hundreds of chimeric BTK molecules and measured their fitness using a high-throughput assay in T and B cells. Surprisingly, many SH2 domains increased fitness when substituted into BTK. Analysis of one set of chimeric proteins demonstrated that the increase in fitness stems from the ability of the substituted SH2 domains to disrupt BTK autoinhibition while maintaining phosphotyrosine targeting. These results reveal the importance of distributed interactions between the SH2 and kinase domains of BTK in stabilizing the inhibitory conformation, and suggest that the specialized latching mechanisms in Src and Abl kinases may be later evolutionary refinements.

biochemistry↗

bioRxiv2024

Autophosphorylation of the oncogenic protein TEL-ABL confers resistance to the allosteric ABL inhibitor asciminib

Chromosomal translocations that fuse the ABL1 gene to BCR and TEL cause human leukemias. Oligomerization and the loss of an inhibitory myristoylation modification lead to unregulated kinase activity of the BCR-ABL and TEL-ABL fusion proteins. ATP-competitive ABL inhibitors, such as imatinib and ponatinib, are effective against both fusion proteins. We discovered that asciminib, an allosteric inhibitor of BCR-ABL that binds to the myristoyl binding site in the ABL kinase domain, is >2000-fold less potent against TEL-ABL than BCR-ABL in cell-growth assays. This is surprising because the ABL components of the two fusion proteins, including the asciminib binding sites, have identical sequence. We deleted a short helical segment in the ABL kinase domain that closes over asciminib when it is bound. This deletion results in asciminib resistance in BCR-ABL, but has no effect on TEL-ABL, suggesting that the native autoinhibitory mechanism that asciminib engages in BCR-ABL is disrupted in TEL-ABL. We show, using mammalian cell expression and single-molecule microscopy, that BCR-ABL is mainly dimeric while TEL-ABL forms higher-order oligomers. Oligomerization can promote trans-autophosphorylation of ABL, and we find that a regulatory phosphorylation site in the SH3 domain of ABL (Tyr 89) is highly phosphorylated in TEL-ABL. This phosphorylation is expected to disassemble the autoinhibited conformation of ABL, thereby preventing asciminib binding. We show that TEL-ABL is intrinsically susceptible to inhibition by asciminib, but that increased phosphorylation results in resistance. Our results demonstrate that different ABL fusion proteins can have dramatically different responses to allosteric inhibitors due to differential phosphorylation. One Sentence SummaryWhen TEL-ABL is phosphorylated, it is insensitive to asciminib. However, when TEL-ABL is dephosphorylated by a phosphatase, asciminib sensitivity is restored.

biochemistry↗

bioRxiv2024

Ligand-induced CaMKIIα hub Trp403 flip, hub domain stacking and kinase inhibition

{gamma}-Hydroxybutyric acid (GHB) analogs are small molecules that bind competitively to a specific cavity in the oligomeric CaMKII hub domain. Binding affects conformation and stability of the hub domain, which may explain the neuroprotective action of some of these compounds. Here, we describe molecular details of interaction of the larger-type GHB analog 2-(6-(4-chlorophenyl)imidazo[1,2-b]pyridazine-2-yl)acetic acid (PIPA). Like smaller-type analogs, PIPA binding to the CaMKII hub domain promoted thermal stability. PIPA additionally inhibited CaMKII kinase activity by reducing CaM sensitivity. A high-resolution X-ray crystal structure of a stabilized CaMKII (6x mutant) hub construct revealed details of the binding mode of PIPA, which involved outward placement of tryptophan 403 (Trp403), a central residue in a flexible loop close to the upper hub cavity. Small-angle X-ray scattering (SAXS) solution structures and mass photometry of the CaMKII wildtype hub domain in the presence of PIPA revealed a high degree of ordered self-association (stacks of CaMKII hub domains). This stacking neither occurred with the smaller compound 3-hydroxycyclopent-1-enecarboxylic acid (HOCPCA), nor when Trp403 was replaced with leucine (W403L). Additionally, CaMKII W403L hub was stabilized to a larger extent by PIPA compared to CaMKII hub wildtype, indicating that loop flexibility is important for holoenzyme stability. Thus, we propose that ligand-induced outward placement of Trp403 by PIPA, which promotes an unforeseen mechanism of hub domain stacking, may be involved in the observed reduction in CaMKII kinase activity. Altogether, this sheds new light on allosteric regulation of CaMKII activity via the hub domain.

biophysics↗

bioRxiv2023

A Fluorescence-Based Sensor for Calibrated Measurement of Protein Kinase Stability in Live Cells

Oncogenic mutations can destabilize signaling proteins, resulting in increased or unregulated activity. Thus, there is considerable interest in mapping the relationship between mutations and the stability of proteins, to better understand the consequences of oncogenic mutations and potentially inform the development of new therapeutics. Here, we develop a tool to study protein-kinase stability in live mammalian cells and the effects of the HSP90 chaperone system on the stability of these kinases. We monitor the fluorescence of kinases fused to a fluorescent protein relative to that of a co-expressed reference fluorescent protein. We used this tool to study the dependence of Src- and Raf-family kinases on the HSP90 system. We demonstrate that this sensor reports on destabilization induced by oncogenic mutations in these kinases. We also show that Src-homology 2 (SH2) and Src-homology 3 (SH3) domains, which are required for autoinhibition of Src-family kinases, stabilize these kinase domains in the cell. Our expression-calibrated sensor enables the facile characterization of the effects of mutations and small-molecule drugs on protein-kinase stability.

biochemistry↗

bioRxiv2023

Adaptive capacity of a DNA polymerase clamp-loader ATPase complex

The ability of mutations to facilitate adaptation is central to evolution. To understand how mutations can lead to functional adaptation in a complex molecular machine, we created a defective version of the T4 clamp-loader complex, which is essential for DNA replication. This variant, which is [~]5000-fold less active than the wildtype, was made by replacing the catalytic domains with those from another phage. A directed-evolution experiment revealed that multiple substitutions to a single negatively-charged residue in the chimeric clamp loader - Asp 86 - restore fitness to within [~]20-fold of wildtype. These mutations remove an adventitious electrostatic repulsive interaction between Asp 86 and the sliding clamp. Deep mutagenesis shows that the reduced fitness of the chimeric clamp loader is compensated for by lysine and arginine substitutions of several DNA-proximal residues in the clamp loader or the sliding clamp. Thus, the fitness decrease of the chimeric clamp loader is caused by a reduction in affinity between the clamp loader and the clamp. Our results demonstrate that there is a latent capacity for increasing affinity of the clamp loader for DNA and the sliding clamp, such that even single point mutations can readily compensate for the loss of function due to suboptimal interactions elsewhere.

biochemistry↗

bioRxiv2023

Differential Requirement for Dimerization of the Membrane-Binding PH-TH Module of BTK in B cells and T cells

Brutons tyrosine kinase (BTK) is a major drug target in immune cells. The membrane-binding pleckstrin-homology and tec-homology (PH-TH) domains of BTK are required for signaling. In vitro, dimerization of the PH-TH module strongly stimulates BTK kinase activity. Whether BTK dimerizes in cells via the PH-TH module, and whether this dimerization is necessary for signaling, is unknown. To address this question, we developed high-throughput mutagenesis assays for BTK function in B cells and T cells. We measured the fitness costs for thousands of point mutations in the PH-TH module and kinase domain, allowing us to assess whether dimerization of the PH-TH module and BTK kinase activity are necessary for function. In Ramos B cells we find that neither PH-TH dimerization nor kinase activity is required for BTK signaling. Instead, in Ramos cells, BTK signaling is enhanced by mutations in the PH-TH module that increase membrane adsorption, even at the cost of reduced PH-TH dimerization. In contrast, in Jurkat T cells, we find that BTK signaling depends on both PH-TH dimerization and kinase activity. Evolutionary analysis shows that BTK proteins in fish and lower organisms, like all Tec kinases other than BTK, lack PH-TH dimerization but have active kinase domains. Thus, PH-TH dimerization is not intrinsically required for Tec-kinase activity, and is a special feature that evolved to exert stricter regulatory control on BTK kinase activity as adaptive immune systems gained increased complexity.

biochemistry↗

bioRxiv2022

Crystal Structure of the kinase domain of a receptor tyrosine kinase from a choanoflagellate, Monosiga brevicollis

Genomic analysis of the unicellular choanoflagellate, Monosiga brevicollis (MB), revealed the remarkable presence of cell signaling and adhesion protein domains that are characteristically associated with metazoans. Strikingly, receptor tyrosine kinases, one of the most critical elements of signal transduction and communication in metazoans, are present in choanoflagellates. We determined the crystal structure at 1.95 [A] resolution of the kinase domain of the M. brevicollis receptor tyrosine kinase C8 (RTKC8, a member of the choanoflagellate receptor tyrosine kinase C family) bound to the kinase inhibitor staurospaurine. The chonanoflagellate kinase domain is closely related in sequence to mammalian tyrosine kinases ([~] 40% sequence identity to the human Ephrin kinase domain EphA3) and, as expected, has the canonical protein kinase fold. The kinase is structurally most similar to human Ephrin (EphA5), even though the extracellular sensor domain is completely different from that of Ephrin. The RTKC8 kinase domain is in an active conformation, with two staurosporine molecules bound to the kinase, one at the active site and another at the peptide-substrate binding site. We show that the RTKC8 kinase domain can phosphorylate tyrosine residues in peptides from its C-terminal tail segment.

biochemistry↗

bioRxiv2022

Stimulation of the catalytic activity of the tyrosine kinase Btk by the adaptor protein Grb2

The Tec-family kinase Btk contains a lipid-binding Pleckstrin homology and Tec homology (PH-TH) module connected by a proline-rich linker to a "Src module", an SH2-SH3-kinase unit also found in Src-family kinases and Abl. We showed previously that Btk is activated by PH-TH dimerization, which is triggered on membranes by the phosphatidyl inositol phosphate PIP3, or in solution by hexakisinositol phosphate (IP6) (Wang et al. 2015, https://doi.org/10.7554/eLife.06074). We now report that the ubiquitous adaptor protein growth-factor-receptor-bound protein 2 (Grb2) binds to and substantially increases the activity of PIP3-bound Btk on membranes. Using reconstitution on supported-lipid bilayers, we find that Grb2 can be recruited to membrane-bound Btk through interaction with the proline-rich linker in Btk. This interaction requires intact Grb2, containing both SH3 domains and the SH2 domain, but does not require that the SH2 domain be able to bind phosphorylated tyrosine residues - thus Grb2 bound to Btk is free to interact with scaffold proteins via the SH2 domain. We show that the Grb2-Btk interaction recruits Btk to scaffold-mediated signaling clusters in reconstituted membranes. Our findings indicate that PIP3-mediated dimerization of Btk does not fully activate Btk, and that Btk adopts an autoinhibited state at the membrane that is released by Grb2.

molecular biology↗

bioRxiv2022

Nanomolar inhibition of SARS-CoV-2 infection by an unmodified peptide targeting the pre-hairpin intermediate of the spike protein

Variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) challenge currently available COVID-19 vaccines and monoclonal antibody therapies through epitope change on the receptor binding domain of the viral spike glycoprotein. Hence, there is a specific urgent need for alternative antivirals that target processes less likely to be affected by mutation, such as the membrane fusion step of viral entry into the host cell. One such antiviral class includes peptide inhibitors which block formation of the so-called HR1HR2 six-helix bundle of the SARS-CoV-2 spike (S) protein and thus interfere with viral membrane fusion. Here we performed structural studies of the HR1HR2 bundle, revealing an extended, well-folded N-terminal region of HR2 that interacts with the HR1 triple helix. Based on this structure, we designed an extended HR2 peptide that achieves single-digit nanomolar inhibition of SARS-CoV-2 in cell-based fusion, VSV-SARS-CoV-2 chimera, and authentic SARS-CoV-2 infection assays without the need for modifications such as lipidation or chemical stapling. The peptide also strongly inhibits all major SARS-CoV-2 variants to date. This extended peptide is ~100-fold more potent than all previously published short, unmodified HR2 peptides, and it has a very long inhibition lifetime after washout in virus infection assays, suggesting that it targets a pre-hairpin intermediate of the SARS-CoV-2 S protein. Together, these results suggest that regions outside the HR2 helical region may offer new opportunities for potent peptide-derived therapeutics for SARS-CoV-2 and its variants, and even more distantly related viruses, and provide further support for the pre-hairpin intermediate of the S protein. Significance StatementSARS-CoV-2 infection requires fusion of viral and host membranes, mediated by the viral spike glycoprotein (S). Due to the importance of viral membrane fusion, S has been a popular target for developing vaccines and therapeutics. We discovered a simple peptide that inhibits infection by all major variants of SARS-CoV-2 with nanomolar efficacies. In marked contrast, widely used shorter peptides that lack a key N-terminal extension are about 100 x less potent than this peptide. Our results suggest that a simple peptide with a suitable sequence can be a potent and cost-effective therapeutic against COVID-19 and they provide new insights at the virus entry mechanism.

microbiology↗

bioRxiv2022

Saturation mutagenesis of a predicted ancestral Syk-family kinase

Many tyrosine kinases cannot be expressed readily in E. coli, limiting facile production of these proteins for biochemical experiments. We used ancestral sequence reconstruction to generate a spleen tyrosine kinase (Syk) variant that can be expressed in bacteria and purified in soluble form, unlike the human members of this family (Syk and ZAP-70). The catalytic activity, substrate specificity, and regulation by phosphorylation of this Syk variant are similar to the corresponding properties of human Syk and ZAP-70. Taking advantage of the ability to express this novel Syk-family kinase in bacteria, we developed a two-hybrid assay that couples the growth of E.coli in the presence of an antibiotic to successful phosphorylation of a bait peptide by the kinase. Using this assay, we screened a site-saturation mutagenesis library of the kinase domain of this reconstructed Syk-family kinase. Sites of loss-of-function mutations identified in the screen correlate well with residues established previously as critical to function and/or structure in protein kinases. We also identified activating mutations in the regulatory hydrophobic spine and activation loop, which are within key motifs involved in kinase regulation. Strikingly, one mutation in an ancestral Syk-family variant increases the soluble expression of the protein by 75-fold. Thus, through ancestral sequence reconstruction followed by deep mutational scanning, we have generated Syk-family kinase variants that can be expressed in bacteria with very high yield.

biochemistry↗

bioRxiv2022

A Saturation-Mutagenesis Analysis of the Interplay Between Stability and Activation in Ras

Cancer mutations in Ras occur predominantly at three hotspots: Gly 12, Gly 13, and Gln 61. Previously, we reported that deep mutagenesis of H-Ras using a bacterial assay identified many other activating mutations (Bandaru et al. eLife, 2017). We now show that the results of saturation mutagenesis of H-Ras in mammalian Ba/F3 cells correlate well with results of bacterial experiments in which H-Ras or K-Ras are co-expressed with a GTPase-activating protein (GAP). The prominent cancer hotspots are not dominant in the Ba/F3 data. We used the bacterial system to mutagenize Ras constructs of different stabilities and discovered a feature that distinguishes the cancer hotspots. While mutations at the cancer hotspots activate Ras regardless of construct stability, mutations at lower-frequency sites (e.g., at Val 14 or Asp 119) can be activating or deleterious, depending on the stability of the Ras construct. We characterized the dynamics of three non-hotspot activating Ras mutants by using NMR to monitor hydrogen-deuterium exchange (HDX). These mutations result in global increases in HDX rates, consistent with the destabilization of Ras. An explanation for these observations is that mutations that destabilize Ras increase nucleotide dissociation rates, enabling activation by spontaneous nucleotide exchange. A further stability decrease can lead to insufficient levels of folded Ras - and subsequent loss of function. In contrast, the cancer hotspot mutations are mechanism-based activators of Ras that interfere directly with the action of GAPs. Our results demonstrate the importance of GAP surveillance and protein stability in determining the sensitivity of Ras to mutational activation.

biophysics↗

bioRxiv2021

A two-component protein condensate of EGFR and Grb2 regulates Ras activation at the membrane

We reconstitute a phosphotyrosine-mediated protein condensation phase transition of the [~]200 residue cytoplasmic tail of the epidermal growth factor receptor (EGFR) and the adaptor protein, Grb2, on a membrane surface. The phase transition depends on phosphorylation of the EGFR tail, which recruits Grb2, and the dimerization of Grb2, which provides the crosslinking element for condensation with EGFR. The Grb2 Y160 residue plays a structurally critical role in dimer formation, and phosphorylation or mutation of Y160 prevents EGFR:Grb2 condensation. By extending the reconstitution experiment to include the guanine nucleotide exchange factor, SOS, and its substrate Ras, we further find that EGFR condensation controls the ability of SOS to activate Ras. These results identify an EGFR:Grb2 protein condensation phase transition as a regulator of signal propagation from EGFR to the MAPK pathway.

biophysics↗

bioRxiv2021

Differences in the dynamics of the tandem-SH2 modules of the Syk and ZAP-70 tyrosine kinases

The catalytic activity of Syk-family tyrosine kinases is regulated by a tandem-SH2 module (tSH2 module). In the autoinhibited state, this module adopts a conformation which stabilizes an inactive conformation of the kinase domain. The binding of the tSH2 module to doubly-phosphorylated tyrosine-containing motifs necessitates a conformational change, thereby relieving kinase inhibition and promoting activation. We determined the crystal structure of the isolated tSH2 module of Syk and find, in contrast to ZAP-70, that its conformation more closely resembles that of the peptide-bound state, rather than the autoinhibited state. Hydrogen-deuterium exchange by mass spectrometry, as well as molecular dynamics simulations, reveal that the dynamics of the tSH2 modules of Syk and ZAP-70 differ, with most of these differences occurring in the C-terminal SH2 domain. Our data suggest that the conformational landscapes of the tSH2 modules in Syk and ZAP-70 have been tuned differently, such that the auto-inhibited conformation of the Syk tSH2 module is less stable. This feature of Syk likely contributes to its ability to more readily escape autoinhibition when compared to ZAP-70, consistent with tighter control of downstream signaling pathways in T cells.

biochemistry↗

bioRxiv2021

Allosteric communication in DNA polymerase clamp loaders relies on a critical hydrogen-bonded junction

Clamp loaders are AAA+ ATPases that load sliding clamps onto DNA. We mapped the mutational sensitivity of the T4 bacteriophage sliding clamp and clamp loader by deep mutagenesis, and found that residues not involved in catalysis or binding display remarkable tolerance to mutation. An exception is a glutamine residue in the AAA+ module (Gln 118) that is not located at a catalytic or interfacial site. Gln 118 forms a hydrogen-bonded junction in a helical unit that we term the central coupler, because it connects the catalytic centers to DNA and the sliding clamp. A suppressor mutation indicates that hydrogen bonding in the junction is important, and molecular dynamics simulations reveal that it maintains rigidity in the central coupler. The glutamine-mediated junction is preserved in diverse AAA+ ATPases, suggesting that a connected network of hydrogen bonds that links ATP molecules is an essential aspect of allosteric communication in these proteins.

biophysics↗