Mechanism Of Allosteric Regulation Research Articles

The mitochondrial calcium uniporter: Balancing tumourigenic and anti-tumourigenic responses.

Increased malignancy and poor treatability associated with solid tumour cancers have commonly been attributed to mitochondrial calcium (Ca2+) dysregulation. The mitochondrial Ca2+ uniporter complex (mtCU) is the predominant mode of Ca2+ uptake into the mitochondrial matrix. The main components of mtCU are the pore-forming mitochondrial Ca2+ uniporter (MCU) subunit, MCU dominant-negative beta (MCUb) subunit, essential MCU regulator (EMRE) and the gatekeeping mitochondrial Ca2+ uptake 1 and 2 (MICU1 and MICU2) proteins. In this review, we describe mtCU-mediated mitochondrial Ca2+ dysregulation in solid tumour cancer types, finding enhanced mtCU activity observed in colorectal cancer, breast cancer, oral squamous cell carcinoma, pancreatic cancer, hepatocellular carcinoma and embryonal rhabdomyosarcoma. By contrast, decreased mtCU activity is associated with melanoma, whereas the nature of mtCU dysregulation remains unclear in glioblastoma. Furthermore, we show that numerous polymorphisms associated with cancer may alter phosphorylation sites on the pore forming MCU and MCUb subunits, which cluster at interfaces with EMRE. We highlight downstream/upstream biomolecular modulators of MCU and MCUb that alter mtCU-mediated mitochondrial Ca2+ uptake and may be used as biomarkers or to aid in the development of novel cancer therapeutics. Additionally, we provide an overview of the current small molecule inhibitors of mtCU that interact with the Asp residue of the critical Asp-Ile-Met-Glu motif or through other allosteric regulatory mechanisms to block Ca2+ permeation. Finally, we describe the relationship between MCU- and MCUb-mediating microRNAs and mitochondrial Ca2+ uptake that should be considered in the discovery of new treatment approaches for cancer.

Allosteric Regulation and Inhibition of Coronavirus 3CLpro Revealed by HDX-MS.

Coronavirus (CoV) infections have caused contagious and fatal respiratory diseases in humans worldwide. CoV 3-chymotrypsin-like proteases (3CLpro or Mpro) play an important role in viral maturation, and maintenance of their dimeric conformation is crucial for viral activity. Therefore, allosterically regulated dimerization of 3CLpro can be employed as a drug development target. Here, we investigated the allosteric regulatory mechanism of 3CLpro dimerization by using hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS) technology. We found that the FLAG tag directly coupled to the N-finger of 3CLpro significantly increased HDX kinetics at the dimer interface, and 3CLpro transformed from a dimer to a monomer. The 3CLpro mutants of SARS-CoV-2, which are monomeric, also exhibited increased deuterium exchange. Binding of the allosteric inhibitor Gastrodenol to most betacoronavirus 3CLpros led to increased allosteric deuterium exchange, resulting in the monomeric conformation of the CoV 3CLpro upon binding. Molecular dynamics (MD) simulation analysis further indicated the molecular mechanism of action of Gastrodenol on CoV 3CLpro: binding of Gastrodenol to SARS-CoV-2 3CLpro destroyed the hydrogen bond in the dimer interface. These results suggest that Gastrodenol may be a potential broad-spectrum anti-betacoronavirus drug.

Exploring Binding Pockets in the Conformational States of the SARS-CoV-2 Spike Trimers for the Screening of Allosteric Inhibitors Using Molecular Simulations and Ensemble-Based Ligand Docking.

Understanding mechanisms of allosteric regulation remains elusive for the SARS-CoV-2 spike protein, despite the increasing interest and effort in discovering allosteric inhibitors of the viral activity and interactions with the host receptor ACE2. The challenges of discovering allosteric modulators of the SARS-CoV-2 spike proteins are associated with the diversity of cryptic allosteric sites and complex molecular mechanisms that can be employed by allosteric ligands, including the alteration of the conformational equilibrium of spike protein and preferential stabilization of specific functional states. In the current study, we combine conformational dynamics analysis of distinct forms of the full-length spike protein trimers and machine-learning-based binding pocket detection with the ensemble-based ligand docking and binding free energy analysis to characterize the potential allosteric binding sites and determine structural and energetic determinants of allosteric inhibition for a series of experimentally validated allosteric molecules. The results demonstrate a good agreement between computational and experimental binding affinities, providing support to the predicted binding modes and suggesting key interactions formed by the allosteric ligands to elicit the experimentally observed inhibition. We establish structural and energetic determinants of allosteric binding for the experimentally known allosteric molecules, indicating a potential mechanism of allosteric modulation by targeting the hinges of the inter-protomer movements and blocking conformational changes between the closed and open spike trimer forms. The results of this study demonstrate that combining ensemble-based ligand docking with conformational states of spike protein and rigorous binding energy analysis enables robust characterization of the ligand binding modes, the identification of allosteric binding hotspots, and the prediction of binding affinities for validated allosteric modulators, which is consistent with the experimental data. This study suggested that the conformational adaptability of the protein allosteric sites and the diversity of ligand bound conformations are both in play to enable efficient targeting of allosteric binding sites and interfere with the conformational changes.

Cryo-EM structures of the human NaS1 and NaDC1 transporters revealed the elevator transport and allosteric regulation mechanism.

The solute carrier 13 (SLC13) family comprises electrogenic sodium ion-coupled anion cotransporters, segregating into sodium ion-sulfate cotransporters (NaSs) and sodium ion-di- and-tricarboxylate cotransporters (NaDCs). NaS1 and NaDC1 regulate sulfate homeostasis and oxidative metabolism, respectively. NaS1 deficiency affects murine growth and fertility, while NaDC1 affects urinary citrate and calcium nephrolithiasis. Despite their importance, the mechanisms of substrate recognition and transport remain insufficiently characterized. In this study, we determined the cryo-electron microscopy structures of human NaS1, capturing inward-facing and combined inward-facing/outward-facing conformations within a dimer both in apo and sulfate-bound states. In addition, we elucidated NaDC1's outward-facing conformation, encompassing apo, citrate-bound, and N-(p-amylcinnamoyl) anthranilic acid (ACA) inhibitor-bound states. Structural scrutiny illuminates a detailed elevator mechanism driving conformational changes. Notably, the ACA inhibitor unexpectedly binds primarily anchored by transmembrane 2 (TM2), Loop 10, TM11, and TM6a proximate to the cytosolic membrane. Our findings provide crucial insights into SLC13 transport mechanisms, paving the way for future drug design.

FuncPhos-STR: An integrated deep neural network for functional phosphosite prediction based on AlphaFold protein structure and dynamics

Phosphorylation modifications play important regulatory roles in most biological processes. However, the functional assignment for the vast majority of the identified phosphosites remains a major challenge. Here, we provide a deep learning framework named FuncPhos-STR as an online resource, for functional prediction and structural visualization of human proteome-level phosphosites. Based on our reported FuncPhos-SEQ framework, which was built by integrating phosphosite sequence evolution and protein-protein interaction (PPI) information, FuncPhos-STR was developed by further integrating the structural and dynamics information on AlphaFold protein structures. The characterized structural topology and dynamics features underlying functional phosphosites emphasized their molecular mechanism for regulating protein functions. By integrating the structural and dynamics, sequence evolutionary, and PPI network features from protein different dimensions, FuncPhos-STR has advantage over other reported models, with the best AUC value of 0.855. Using FuncPhos-STR, the phosphosites inside the pocket regions are accessible to higher functional scores, theoretically supporting their potential regulatory mechanism. Overall, FuncPhos-STR would accelerate the functional identification of huge unexplored phosphosites, and facilitate the elucidation of their allosteric regulation mechanisms. The web server of FuncPhos-STR is freely available at http://funcptm.jysw.suda.edu.cn/str.

Widespread alteration of protein autoinhibition in human cancers

Autoinhibition is a prevalent allosteric regulatory mechanism in signaling proteins. Reduced autoinhibition underlies the tumorigenic effect of some known cancer drivers, but whether autoinhibition is altered generally in cancer remains elusive. Here, we demonstrate that cancer-associated missense mutations, in-frame insertions/deletions, and fusion breakpoints are enriched within inhibitory allosteric switches (IASs) across all cancer types. Selection for IASs that are recurrently mutated in cancers identifies established and unknown cancer drivers. Recurrent missense mutations in IASs of these drivers are associated with distinct, cancer-specific changes in molecular signaling. For the specific case of PPP3CA, the catalytic subunit of calcineurin, we provide insights into the molecular mechanisms of altered autoinhibition by cancer mutations using biomolecular simulations, and demonstrate that such mutations are associated with transcriptome changes consistent with increased calcineurin signaling. Our integrative study shows that autoinhibition-modulating genetic alterations are positively selected for by cancer cells.

Dissecting the Allosteric Fine-Tuning of Enzyme Catalysis.

Fully understanding the mechanism of allosteric regulation in biomolecules requires separating and examining all of the involved factors. In enzyme catalysis, allosteric effector binding shifts the structure and dynamics of the active site, leading to modified energetic (e.g., energy barrier) and dynamical (e.g., diffusion coefficient) factors underlying the catalyzed reaction rate. Such modifications can be subtle and dependent on the type of allosteric effector, representing a fine-tuning of protein function. The microscopic description of allosteric regulation at the level of function-dictating factors has prospective applications in fundamental and pharmaceutical sciences, which is, however, largely missing so far. Here, we characterize the allosteric fine-tuning of enzyme catalysis, using human Pin1 as an example, by performing more than half-millisecond all-atom molecular dynamics simulations. Changes of reaction kinetics and the dictating factors, including the free energy surface along the reaction coordinate and the diffusion coefficient of the reaction dynamics, under various enzyme and allosteric effector binding conditions are examined. Our results suggest equal importance of the energetic and dynamical factors, both of which can be modulated allosterically, and the combined effect determines the final allosteric output. We also reveal the potential dynamic basis for allosteric modulation using an advanced statistical technique to detect function-related conformational dynamics. Methods developed in this work can be applied to other allosteric systems.

The Allosteric Regulation of Β-Ureidopropionase Depends on Fine-Tuned Stability of Active-Site Loops and Subunit Interfaces.

The activity of β-ureidopropionase, which catalyses the last step in the degradation of uracil, thymine, and analogous antimetabolites, is cooperatively regulated by the substrate and product of the reaction. This involves shifts in the equilibrium of the oligomeric states of the enzyme, but how these are achieved and result in changes in enzyme catalytic competence has yet to be determined. Here, the regulation of human β-ureidopropionase was further explored via site-directed mutagenesis, inhibition studies, and cryo-electron microscopy. The active-site residue E207, as well as H173 and H307 located at the dimer-dimer interface, are shown to play crucial roles in enzyme activation. Dimer association to larger assemblies requires closure of active-site loops, which positions the catalytically crucial E207 stably in the active site. H173 and H307 likely respond to ligand-induced changes in their environment with changes in their protonation states, which fine-tunes the active-site loop stability and the strength of dimer-dimer interfaces and explains the previously observed pH influence on the oligomer equilibrium. The correlation between substrate analogue structure and effect on enzyme assembly suggests that the ability to favourably interact with F205 may distinguish activators from inhibitors. The cryo-EM structure of human β-ureidopropionase assembly obtained at low pH provides first insights into the architecture of its activated state. and validates our current model of the allosteric regulation mechanism. Closed entrance loop conformations and dimer-dimer interfaces are highly conserved between human and fruit fly enzymes.

Unraveling the mechanism of ceftaroline-induced allosteric regulation in penicillin-binding protein 2a: insights for novel antibiotic development against methicillin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) acquires high-level resistance against β-lactam antibiotics by expressing penicillin-binding protein 2a (PBP2a). PBP2a is a cell wall-synthesizing protein whose closed active site exhibits a reduced binding affinity toward β-lactam antibiotics. Ceftaroline (CFT), a fifth-generation cephalosporin, can effectively inhibit the PBP2a activity by binding to an allosteric site to trigger the active site opening, allowing a second CFT to access the active site. However, the essential mechanism behind the allosteric behavior of PBP2a remains unclear. Herein, computational simulations are employed to elucidate how CFT allosterically regulates the conformation and dynamics of the active site of PBP2a. While CFT stabilizes the allosteric domain surrounding it, it simultaneously enhances the dynamics of the catalytic domain. Specifically, the study successfully captured the opening process of the active pocket in the allosteric CFT-bound systems and discovered that CFT alters the potential signal-propagating pathways from the allosteric site to the active site. These findings reveal the implied mechanism of the CFT-mediated allostery in PBP2a and provide new insights into dual-site drug design or combination therapy against MRSA targeting PBP2a.

Biophysical Mechanism of Allosteric Regulation of Actin Capping Protein

Actin capping protein (CP) can be regulated by steric and allosteric mechanisms. The molecular mechanism of the allosteric regulation at a biophysical level includes linkage between the binding sites for three ligands: F-actin, Capping-Protein-Interacting (CPI) motifs, and V-1/myotrophin, based on biochemical functional studies and solvent accessibility experiments. Here, we investigated the mechanism of allosteric regulation at the atomic level using single-molecule Förster resonance energy transfer (FRET) and molecular dynamics (MD) to assess the conformational and structural dynamics of CP in response to linked-binding site ligands. In the absence of ligand, both single-molecule FRET and MD revealed two distinct conformations of CP in solution; previous crystallographic studies revealed only one. Interaction with CPI-motif peptides induced conformations within CP that bring the cap and stalk closer, while interaction with V-1 moves them away from one another. Comparing CPI-motif peptides from different proteins, we identified variations in CP conformations and dynamics that are specific to each CPI motif. MD simulations for CP alone and in complex with a CPI motif and V-1 reveal atomistic details of the conformational changes. Analysis of the interaction of CP with wild-type (wt) and chimeric CPI-motif peptides using single-molecule FRET, isothermal calorimetry (ITC) and MD simulation indicated that conformational and affinity differences are intrinsic to the C-terminal portion of the CPI motif. We conclude that allosteric regulation of CP involves changes in conformation that disseminate across the protein to link distinct binding-site functions. Our results provide novel insights into the biophysical mechanism of the allosteric regulation of CP.

Metabolic flux phenotyping of secondary metabolism in cyanobacteria.

Cyanobacteria generate energy from photosynthesis and produce various secondary metabolites with diverse commercial and pharmaceutical applications. Unique metabolic and regulatory pathways in cyanobacteria present new challenges for researchers to enhance their product yields, titers, and rates. Therefore, further advancements are critically needed to establish cyanobacteria as a preferred bioproduction platform. Metabolic flux analysis (MFA) quantitatively determines the intracellular flows of carbon within complex biochemical networks, which elucidate the control of metabolic pathways by transcriptional, translational, and allosteric regulatory mechanisms. The emerging field of systems metabolic engineering (SME) involves the use of MFA and other omics technologies to guide the rational development of microbial production strains. This review highlights the potential of MFA and SME to optimize the production of cyanobacterial secondary metabolites and discusses the technical challenges that lie ahead.

State-of-the-art in engineering small molecule biosensors and their applications in metabolic engineering

Genetically encoded biosensors are crucial for enhancing our understanding of how molecules regulate biological systems. Small molecule biosensors, in particular, help us understand the interaction between chemicals and biological processes. They also accelerate metabolic engineering by increasing screening throughput and eliminating the need for sample preparation through traditional chemical analysis. Additionally, they offer significantly higher spatial and temporal resolution in cellular analyte measurements. In this review, we discuss recent progress in in vivo biosensors and control systems—biosensor-based controllers—for metabolic engineering. We also specifically explore protein-based biosensors that utilize less commonly exploited signaling mechanisms, such as protein stability and induced degradation, compared to more prevalent transcription factor and allosteric regulation mechanism. We propose that these lesser-used mechanisms will be significant for engineering eukaryotic systems and slower-growing prokaryotic systems where protein turnover may facilitate more rapid and reliable measurement and regulation of the current cellular state. Lastly, we emphasize the utilization of cutting-edge and state-of-the-art techniques in the development of protein-based biosensors, achieved through rational design, directed evolution, and collaborative approaches.

Neuronal and astrocyte NCX isoform/splice variants: How do they participate in Na+ and Ca2+ signalling?

NCX1, NCX2, and NCX3 gene isoforms and their splice variants are characteristically expressed in different regions of the brain. The tissue-specific splice variants of NCX1–3 isoforms show specific expression profiles in neurons and astrocytes, whereas the relevant NCX isoform/splice variants exhibit diverse allosteric modes of Na+- and Ca2+-dependent regulation. In general, overexpression of NCX1–3 genes leads to neuroprotective effects, whereas their ablation gains the opposite results. At this end, the partial contributions of NCX isoform/splice variants to neuroprotective effects remain unresolved. The glutamate-dependent Na+ entry generates Na+ transients (in response to neuronal cell activities), whereas the Na+-driven Ca2+ entry (through the reverse NCX mode) raises Ca2+ transients. This special mode of signal coupling translates Na+ transients into the Ca2+ signals while being a part of synaptic neurotransmission. This mechanism is of general interest since disease-related conditions (ischemia, metabolic stress, and stroke among many others) trigger Na+ and Ca2+ overload with deadly outcomes of downstream apoptosis and excitotoxicity. The recently discovered mechanisms of NCX allosteric regulation indicate that some NCX variants might play a critical role in the dynamic coupling of Na+-driven Ca2+ entry. In contrast, the others are less important or even could be dangerous under altered conditions (e.g., metabolic stress). This working hypothesis can be tested by applying advanced experimental approaches and highly focused computational simulations. This may allow the development of structure-based blockers/activators that can selectively modulate predefined NCX variants to lessen the life-threatening outcomes of excitotoxicity, ischemia, apoptosis, metabolic deprivation, brain injury, and stroke.

Chronic cough relief by allosteric modulation of P2X3 without taste disturbance

P2X receptors are cation channels that sense extracellular ATP. Many therapeutic candidates targeting P2X receptors have begun clinical trials or acquired approval for the treatment of refractory chronic cough (RCC) and other disorders. However, the present negative allosteric modulation of P2X receptors is primarily limited to the central pocket or the site below the left flipper domain. Here, we uncover a mechanism of allosteric regulation of P2X3 in the inner pocket of the head domain (IP-HD), and show that the antitussive effects of quercetin and PSFL2915 (our nM-affinity P2X3 inhibitor optimized based on quercetin) on male mice and guinea pigs were achieved by preventing allosteric changes of IP-HD in P2X3. While being therapeutically comparable to the newly licensed P2X3 RCC drug gefapixant, quercetin and PSFL2915 do not have an adverse effect on taste as gefapixant does. Thus, allosteric modulation of P2X3 via IP-HD may be a druggable strategy to alleviate RCC.

The regulation of RGLG2-VWA by Ca2+ ions

RGLG2, an E3 ubiquitin ligase in Arabidopsis thaliana, affects hormone signaling and participates in drought regulation. Here, we determined two crystal structures of RGLG2 VWA domain, representing two conformations, open and closed, respectively. The two structures reveal that Ca2+ ions are allosteric regulators of RGLG2-VWA, which adopts open state when NCBS1(Novel Calcium ions Binding Site 1) binds Ca2+ ions and switches to closed state after Ca2+ ions are removed. This mechanism of allosteric regulation is identical to RGLG1-VWA, but distinct from integrin α and β VWA domains. Therefore, our data provide a backdrop for understanding the role of the Ca2+ ions in conformational change of VWA domain. In addition, we found that RGLG2closed, corresponding to low affinity, can bind pseudo-ligand, which has never been observed in other VWA domains.

Cancer-associated somatic mutations in human phosphofructokinase-1 reveal a critical electrostatic interaction for allosteric regulation of enzyme activity.

Metabolic reprogramming, including increased glucose uptake and lactic acid excretion, is a hallmark of cancer. The glycolytic "gatekeeper" enzyme phosphofructokinase-1 (PFK1), which catalyzes the step committing glucose to breakdown, is dysregulated in cancers. While altered PFK1 activity and expression in tumors have been demonstrated, little is known about the effects of cancer-associated somatic mutations. Somatic mutations in PFK1 inform our understanding of allosteric regulation by identifying key amino acid residues involved in the regulation of enzyme activity. Here, we characterized mutations disrupting an evolutionarily conserved salt bridge between aspartic acid and arginine in human platelet (PFKP) and liver (PFKL) isoforms. Using purified recombinant proteins, we showed that disruption of the Asp-Arg pair in two PFK1 isoforms decreased enzyme activity and altered allosteric regulation. We determined the crystal structure of PFK1 to 3.6Å resolution and used molecular dynamic simulations to understand molecular mechanisms of altered allosteric regulation. We showed that PFKP-D564N had a decreased total system energy and changes in the electrostatic surface potential of the effector site. Cells expressing PFKP-D564N demonstrated a decreased rate of glycolysis, while their ability to induce glycolytic flux under conditions of low cellular energy was enhanced compared to cells expressing wild-type PFKP. Taken together, these results suggest that mutations in Arg-Asp pair at the interface of the catalytic-regulatory domains stabilizes the t-state and presents novel mechanistic insight for therapeutic development in cancer.

Allosteric modulation of SHP2: Quest from known to unknown.

Src homology-2 domain-containing protein tyrosine phosphatase-2 (SHP2) is a key regulatory factor in the cell cycle and its activating mutations play an important role in the development of various cancers, making it an important target for antitumor drugs. Due to the highly conserved amino acid sequence and positively charged nature of the active site of SHP2, it is difficult to discover inhibitors with high affinity for the catalytic site of SHP2 and sufficient cell permeability, making it considered an "undruggable" target. However, the discovery of allosteric regulation mechanisms provides new opportunities for transforming undruggable targets into druggable ones. Given the limitations of orthosteric inhibitors, SHP2 allosteric inhibitors have become a more selective and safer research direction. In this review, we elucidate the oncogenic mechanism of SHP2 and summarize the discovery methods of SHP2 allosteric inhibitors, providing new strategies for the design and improvement of SHP2 allosteric inhibitors.

Biochemical functions and structure of Caenorhabditis elegans ZK177.8 protein: Aicardi–Goutières syndrome SAMHD1 dNTPase ortholog

Mutations in SAM domain and HD domain containing protein 1 (SAMHD1) are found in a neuro-developmental disorder, Aicardi-Goutières Syndrome (AGS) and cancers, and SAMHD1, which is a dNTP tri-phosphorylase, was identified as a myeloid specific HIV-1 restriction factor. Here, we characterized the enzymology and structure of a SAMHD1 ortholog of C. elegans, ZK177.8, which also reportedly induces developmental defects upon gene knockdown. We found ZK177.8 protein is a dNTPase allosterically regulated by dGTP. The active site of ZK177.8 recognizes both 2' OH and tri-phosphate moieties of dNTPs, but not base moiety. The dGTP activator induces the formation of the enzymatically active ZK177.8 tetramers, and ZK177.8 protein lowers cellular dNTP levels in a human monocytic cell line. Finally, ZK177.8 tetramers display very similar X-ray crystal structure with human and mouse SAMHD1s except its lack of the canonical SAM domain. This striking conservation in structure, function and allosteric regulatory mechanism for the hydrolysis of the DNA building blocks supports their host developmental roles.

PAR recognition by PARP1 regulates DNA-dependent activities and independently stimulates catalytic activity of PARP1.

Poly(ADP-ribosyl)ation is predominantly catalyzed by Poly(ADP-ribose) polymerase 1 (PARP1) in response to DNA damage, mediating the DNA repair process to maintain genomic integrity. Single-strand (SSB) and double-strand (DSB) DNA breaks are bona fide stimulators of PARP1 activity. However, PAR-mediated PARP1 regulation remains unexplored. Here, we report ZnF3, BRCT, and WGR, hitherto uncharacterized, as PAR reader domains of PARP1. Surprisingly, these domains recognize PARylated protein with a higher affinity compared with PAR but bind with weak or no affinity to DNA breaks as standalone domains. Conversely, ZnF1 and ZnF2 of PARP1 recognize DNA breaks but bind weakly to PAR. In addition, PAR reader domains, together, exhibit a synergy to recognize PAR or PARylated protein. Further competition-binding studies suggest that PAR binding releases DNA from PARP1, and the WGR domain facilitates DNA release. Unexpectedly, PAR showed catalytic stimulation of PARP1 but hampered the DNA-dependent stimulation. Altogether, our work discovers dedicated high-affinity PAR reader domains of PARP1 and uncovers a novel mechanism of allosteric regulation of DNA-dependent and DNA-independent activities of PARP1 by its catalytic product PAR.

Propeptide-Mediated Allosteric Regulation of Xylanase Xyl-1: An Integrated Experimental and Computational Analysis.

Most GH11 family endo-β-1,4-xylanases contain a propeptide region linked to the N-terminal region. The mechanistic basis of this region harboring key regulation information for enzyme function, however, remains poorly understood. We reported an investigation on the allosteric regulation mechanism of the propeptide based on biochemical characterization, molecular dynamics simulations, and evolutionary analysis. We discovered that the mutant of truncated propeptide shows a remarkably increased thermal stability (melting temperature increased by 11.5 °C) and catalytic efficiency (1.7-fold kcat/Km value of wild type). Molecular dynamics simulations reveal that long-range fluctuations in the propeptide lead to a conformational perturbation in the catalytic pocket and the thumb region. The probability of sampling the active conformation during the glycosylation step is reduced (i.e., catalytic efficiency). In-depth sequence analysis indicates that the propeptide has a strong plasticity and degeneration trend, and propeptide truncation experiments of the homologous enzyme XynB validated the feasibility of the truncation strategy. This work reveals the role of GH11 family propeptides in functional regulation and provides a straightforward and practical method to increase the robustness of GH11 family xylanases.