Aberrant DNA N6 -methyladenine incorporation via adenylate kinase 1 is suppressed by ADAL deaminase-dependent 2'-deoxynucleotide pool sanitation.

Intracellular decay of N6 -methyladenine (m6A)-containing RNA potentially induces aberrant N6 -methyl-2'-adenine (6mdA) misincorporation into DNA. Biophysically, misincorporated 6mdA may destabilize the DNA duplex in a manner similar to bona fide methylated 6mdA DNA, thereby affecting DNA replication and transcription. Utilizing heavy stable isotope labeling and ultrasensitive UHPLC-MS/MS assay, we demonstrate that intracellular m6A-RNA decay does not generate free 6mdA species, nor lead to any misincorporated DNA 6mdA in most mammalian cell lines tested, unveiling the existence of a sanitation mechanism that prevents 6mdA misincorporation. Depletion of deaminase ADAL increases the levels of free 6mdA species, concomitant with the presence of DNA-misincorporated 6mdA resulting from intracellular RNA m6A decay, suggesting that ADAL catabolizes 6mdAMP in vivo. Furthermore, we show that the overexpression of adenylate kinase 1 (AK1) promotes 6mdA misincorporation, while AK1 knockdown diminishes 6mdA incorporation, in ADAL-deficient cells. We conclude that ADAL together with other factors (such as MTH1) contributes to 2'-deoxynucleotide pool sanitation in most cells but compromised sanitation (e.g., in NIH3T3 cells) and increased AK1 expression may facilitate aberrant 6mdA incorporation. This sanitation mechanism may provide a framework for the maintenance of the epigenetic 6mdA landscape.

NSD2 drives t(4;14) myeloma cell dependence on adenylate kinase 2 by diverting one-carbon metabolism to the epigenome.

ABSTRACT: Chromosomal translocation (4;14), an adverse prognostic factor in multiple myeloma (MM), drives overexpression of the histone methyltransferase nuclear receptor binding SET domain protein 2 (NSD2). A genome-wide CRISPR screen in MM cells identified adenylate kinase 2 (AK2), an enzyme critical for high-energy phosphate transfer from the mitochondria, as an NSD2-driven vulnerability. AK2 suppression in t(4;14) MM cells decreased nicotinamide adenine dinucleotide phosphate (NADP[H]) critical for conversion of ribonucleotides to deoxyribonucleosides, leading to replication stress, DNA damage, and apoptosis. Driving a large genome-wide increase in chromatin methylation, NSD2 overexpression depletes S-adenosylmethionine, compromising the synthesis of creatine from its precursor, guanidinoacetate. Creatine supplementation restored NADP(H) levels, reduced DNA damage, and rescued AK2-deficient t(4;14) MM cells. As the creatine phosphate shuttle constitutes an alternative means for mitochondrial high-energy phosphate transport, these results indicate that NSD2-driven creatine depletion underlies the hypersensitivity of t(4;14) MM cells to AK2 loss. Furthermore, AK2 depletion in t(4;14) cells impaired protein folding in the endoplasmic reticulum, consistent with impaired use of mitochondrial adenosine triphosphate (ATP). Accordingly, AK2 suppression increased the sensitivity of MM cells to proteasome inhibition. These findings delineate a novel mechanism in which aberrant transfer of carbon to the epigenome creates a metabolic vulnerability, with direct therapeutic implications for t(4;14) MM.

Adenylate kinase 9 is essential for sperm function and male fertility in mammals.

Despite passing routine laboratory tests for semen quality, bulls used in artificial insemination exhibit significant variation in fertility. Routine analysis of fertility data identified a dairy bull with extreme subfertility (10% pregnancy rate). To characterize the subfertility phenotype, a range of in vitro, in vivo, and molecular assays were carried out. Sperm from the subfertile bull exhibited reduced motility and severely reduced caffeine-induced hyperactivation compared to controls. Ability to penetrate the zona pellucida, cleavage rate, cleavage kinetics, and blastocyst yield after IVF or AI were significantly lower than in control bulls. Whole-genome sequencing from semen and RNA sequencing of testis tissue revealed a critical mutation in adenylate kinase 9 (AK9) that impaired splicing, leading to a premature termination codon and a severely truncated protein. Mice deficient in AK9 were generated to further investigate the function of the gene; knockout males were phenotypically indistinguishable from their wild-type littermates but produced immotile sperm that were incapable of normal fertilization. These sperm exhibited numerous abnormalities, including a low ATP concentration and reduced motility. RNA-seq analysis of their testis revealed differential gene expression of components of the axoneme and sperm flagellum as well as steroid metabolic processes. Sperm ultrastructural analysis showed a high percentage of sperm with abnormal flagella. Combined bovine and murine data indicate the essential metabolic role of AK9 in sperm motility and/or hyperactivation, which in turn affects sperm binding and penetration of the zona pellucida. Thus, AK9 has been found to be directly implicated in impaired male fertility in mammals.

The ATPase Fap7 Tests the Ability to Carry Out Translocation-like Conformational Changes and Releases Dim1 during 40S Ribosome Maturation.

Late in their maturation, nascent small (40S) ribosomal subunits bind 60S subunits to produce 80S-like ribosomes. Because of the analogy of this translation-like cycle to actual translation, and because 80S-like ribosomes do not produce any protein, it has been suggested that this represents a quality control mechanism for subunit functionality. Here we use genetic and biochemical experiments to show that the essential ATPase Fap7 promotes formation of the rotated state, a key intermediate in translocation, thereby releasing the essential assembly factor Dim1 from pre-40S subunits. Bypassing this quality control step produces defects in reading frame maintenance. These results show how progress in the maturation cascade is linked to a test for a key functionality of 40S ribosomes: their ability to translocate the mRNA⋅tRNA pair. Furthermore, our data demonstrate for the first time that the translation-like cycle is a quality control mechanism that ensures the fidelity of the cellular ribosome pool.

A novel homozygous nonsense variant of AK7 is associated with multiple morphological abnormalities of the sperm flagella.

RESEARCH QUESTION: Is the novel homozygous nonsense variant of AK7 associated with multiple morphological abnormalities of the sperm flagella (MMAF), a specific type of oligoasthenoteratozoospermia leading to male infertility? DESIGN: Whole-exome sequencing and Sanger sequencing were performed to identify potential gene variants. Immunoblotting and immunofluorescence were applied to confirm the relationship between mutated genes and disease phenotypes. The concentration of reactive oxygen species and the rate of apoptosis were measured to evaluate the mitochondrial function of spermatozoa. Transmission electron microscopy and scanning electron microscopy were employed to observe sperm ultrastructure. RESULTS: A novel homozygous nonsense variant of AK7, c.1153A>T (p. Lys385*), was identified in two infertile siblings with asthenoteratozoospermia through whole-exome sequencing. Both immunoblotting and immunofluorescence assays showed practically complete absence of AK7 in the patient's spermatozoa. Additionally, the individual with the novel AK7 variant exhibited a phenotype characterized by severe oxidative stress and apoptosis caused by mitochondrial metabolic dysfunction of spermatozoa. Notably, remarkable flagellar defects with multiple axonemes in uniflagellate spermatozoa, accompanied by mitochondrial vacuolization, were observed; this has not been reported previously in patients with other AK7 variants. CONCLUSIONS: This study found that a novel identified homozygous nonsense variant of AK7 may be associated with MMAF-related asthenoteratozoospermia. The observed functional associations between mitochondria and sperm flagellar assembly provide evidence for potential mutual regulation between AK7 and flagella-associated proteins during spermatogenesis.

Dynamic allostery can drive cold adaptation in enzymes.

Adaptation of organisms to environmental niches is a hallmark of evolution. One prevalent example is that of thermal adaptation, in which two descendants evolve at different temperature extremes1,2. Underlying the physiological differences between such organisms are changes in enzymes that catalyse essential reactions 3 , with orthologues from each organism undergoing adaptive mutations that preserve similar catalytic rates at their respective physiological temperatures4,5. The sequence changes responsible for these adaptive differences, however, are often at surface-exposed sites distant from the substrate-binding site, leaving the active site of the enzyme structurally unperturbed6,7. How such changes are allosterically propagated to the active site, to modulate activity, is not known. Here we show that entropy-tuning changes can be engineered into distal sites of Escherichia coli adenylate kinase, allowing us to quantitatively assess the role of dynamics in determining affinity, turnover and the role in driving adaptation. The results not only reveal a dynamics-based allosteric tuning mechanism, but also uncover a spatial separation of the control of key enzymatic parameters. Fluctuations in one mobile domain (the LID) control substrate affinity, whereas dynamic attenuation in the other domain (the AMP-binding domain) affects rate-limiting conformational changes that govern enzyme turnover. Dynamics-based regulation may thus represent an elegant, widespread and previously unrealized evolutionary adaptation mechanism that fine-tunes biological function without altering the ground state structure. Furthermore, because rigid-body conformational changes in both domains were thought to be rate limiting for turnover8,9, these adaptation studies reveal a new model for understanding the relationship between dynamics and turnover in adenylate kinase.

An open interface in the pre-80S ribosome coordinated by ribosome assembly factors Tsr1 and Dim1 enables temporal regulation of Fap7.

During their maturation, nascent 40S subunits enter a translation-like quality control cycle, where they are joined by mature 60S subunits to form 80S-like ribosomes. While these assembly intermediates are essential for maturation and quality control, how they form, and how their structure promotes quality control, remains unknown. To address these questions, we determined the structure of an 80S-like ribosome assembly intermediate to an overall resolution of 3.4 Å. The structure, validated by biochemical data, resolves a large body of previously paradoxical data and illustrates how assembly and translation factors cooperate to promote the formation of an interface that lacks many mature subunit contacts but is stabilized by the universally conserved methyltransferase Dim1. We also show how Tsr1 enables this interface by blocking the canonical binding of eIF5B to 40S subunits, while maintaining its binding to 60S. The structure also shows how this interface leads to unfolding of the platform, which allows for temporal regulation of the ATPase Fap7, thus linking 40S maturation to quality control during ribosome assembly.

Differential effect of canagliflozin, a sodium-glucose cotransporter 2 (SGLT2) inhibitor, on slow and fast skeletal muscles from nondiabetic mice.

There has been a concern that sodium-glucose cotransporter 2 (SGLT2) inhibitors could reduce skeletal muscle mass and function. Here, we examine the effect of canagliflozin (CANA), an SGLT2 inhibitor, on slow and fast muscles from nondiabetic C57BL/6J mice. In this study, mice were fed with or without CANA under ad libitum feeding, and then evaluated for metabolic valuables as well as slow and fast muscle mass and function. We also examined the effect of CANA on gene expressions and metabolites in slow and fast muscles. During SGLT2 inhibition, fast muscle function is increased, as accompanied by increased food intake, whereas slow muscle function is unaffected, although slow and fast muscle mass is maintained. When the amount of food in CANA-treated mice is adjusted to that in vehicle-treated mice, fast muscle mass and function are reduced, but slow muscle was unaffected during SGLT2 inhibition. In metabolome analysis, glycolytic metabolites and ATP are increased in fast muscle, whereas glycolytic metabolites are reduced but ATP is maintained in slow muscle during SGLT2 inhibition. Amino acids and free fatty acids are increased in slow muscle, but unchanged in fast muscle during SGLT2 inhibition. The metabolic effects on slow and fast muscles are exaggerated when food intake is restricted. This study demonstrates the differential effects of an SGLT2 inhibitor on slow and fast muscles independent of impaired glucose metabolism, thereby providing new insights into how they should be used in patients with diabetes, who are at a high risk of sarcopenia.

Moving beyond static snapshots: Protein dynamics and the Protein Data Bank.

Proteins are the molecular machines of living systems. Their dynamics are an intrinsic part of their evolutionary selection in carrying out their biological functions. Although the dynamics are more difficult to observe than a static, average structure, we are beginning to observe these dynamics and form sound mechanistic connections between structure, dynamics, and function. This progress is highlighted in case studies from myoglobin and adenylate kinase to the ribosome and molecular motors where these molecules are being probed with a multitude of techniques across many timescales. New approaches to time-resolved crystallography are allowing simple "movies" to be taken of proteins in action, and new methods of mapping the variations in cryo-electron microscopy are emerging to reveal a more complete description of life's machines. The results of these new methods are aided in their dissemination by continual improvements in curation and distribution by the Protein Data Bank and their partners around the world.

Structural Dynamics Support Electrostatic Interactions in the Active Site of Adenylate Kinase.

Electrostatic preorganization as well as structural and dynamic heterogeneity are often used to rationalize the remarkable catalytic efficiency of enzymes. However, they are often presented as incompatible because the generation of permanent electrostatic effects implies that the protein structure remains rigid. Here, we use a metric, electric fields, that can treat electrostatic contributions and dynamics effects on equal footing, for a unique perspective on enzymatic catalysis. We find that the residues that contribute the most to electrostatic interactions with the substrate in the active site of Adenylate Kinase (our working example) are also the most flexible residues. Further, entropy-tuning mutations raise flexibility at the picosecond timescale where more conformations can be visited on short time periods, thereby softening the sharp heterogeneity normally visible at the microsecond timescale.

The energy sensor AMPK orchestrates metabolic and translational adaptation in expanding T helper cells.

The importance of cellular metabolic adaptation in inducing robust T cell responses is well established. However, the mechanism by which T cells link information regarding nutrient supply to clonal expansion and effector function is still enigmatic. Herein, we report that the metabolic sensor adenosine monophosphate-activated protein kinase (AMPK) is a critical link between cellular energy demand and translational activity and, thus, orchestrates optimal expansion of T cells in vivo. AMPK deficiency did not affect T cell fate decision, activation, or T effector cell generation; however, the magnitude of T cell responses in murine in vivo models of T cell activation was markedly reduced. This impairment was global, as all T helper cell subsets were similarly sensitive to loss of AMPK which resulted in reduced T cell accumulation in peripheral organs and reduced disease severity in pathophysiologically as diverse models as T cell transfer colitis and allergic airway inflammation. T cell receptor repertoire analysis confirmed similar clonotype frequencies in different lymphoid organs, thereby supporting the concept of a quantitative impairment in clonal expansion rather than a skewed qualitative immune response. In line with these findings, in-depth metabolic analysis revealed a decrease in T cell oxidative metabolism, and gene set enrichment analysis indicated a major reduction in ribosomal biogenesis and mRNA translation in AMPK-deficient T cells. We, thus, provide evidence that through its interference with these delicate processes, AMPK orchestrates the quantitative, but not the qualitative, manifestation of primary T cell responses in vivo.

Human adenylate kinase 6 regulates WNK1 (with no lysine kinase-1) phosphorylation states and affects ion homeostasis in NT2 cells.

Adenylate kinase 6 (AK6), a nucleus localized phosphotransferase in mammalians, shows ubiquitously expression and broad substrate activity in different tissues and cell types. Although the function of AK6 has been extensively studied in different cancer cell lines, its role in mammalian germline is still unknown. Here we showed that knockdown of AK6 inhibits cell proliferation and promotes cell apoptosis in human testicular carcinoma (NT2 cells). Co-immunoprecipitation experiment and in vitro pull down assay identified WNK1 (with no lysine kinase-1) as one of the AK6 interacting proteins in NT2 cells. Moreover, we found that AK6 regulates the phosphorylation states of WNK1 (Thr60) and affects phosphorylation level of Akt (Ser473) upon hypotonic condition, probably affecting chloride channel and regulating ion transport and homeostasis in NT2 cells and consequently contributing to the decreased cell proliferation rate. In conclusion, AK6 regulates WNK1 phosphorylation states and affects ion homeostasis in NT2 cells. These findings provide new insights into the function of AK6 and WNK1 in human testicular carcinoma. This work also provides foundation for further mechanism study of AK6 in spermatogenesis.

Substrate inhibition imposes fitness penalty at high protein stability.

Proteins are only moderately stable. It has long been debated whether this narrow range of stabilities is solely a result of neutral drift toward lower stability or purifying selection against excess stability-for which no experimental evidence was found so far-is also at work. Here, we show that mutations outside the active site in the essential Escherichia coli enzyme adenylate kinase (Adk) result in a stability-dependent increase in substrate inhibition by AMP, thereby impairing overall enzyme activity at high stability. Such inhibition caused substantial fitness defects not only in the presence of excess substrate but also under physiological conditions. In the latter case, substrate inhibition caused differential accumulation of AMP in the stationary phase for the inhibition-prone mutants. Furthermore, we show that changes in flux through Adk could accurately describe the variation in fitness effects. Taken together, these data suggest that selection against substrate inhibition and hence excess stability may be an important factor determining stability observed for modern-day Adk.

A novel homozygous missense mutation in AK7 causes multiple morphological anomalies of the flagella and oligoasthenoteratozoospermia.

PURPOSE: To identify the genetic causes of multiple morphological anomalies of the flagella (MMAF) and oligoasthenoteratozoospermia (OAT). METHODS: Whole-exome sequencing (WES) was performed on the proband to identify pathogenic mutation for infertility. Western blotting and immunofluorescence analysis detected the expression level and localization of adenylate kinase 7 (AK7). RESULTS: We identified a novel homozygous missense mutation (NM_152327: c.1846G > A; p.E616K) in AK7 in two brothers with MMAF and OAT from a consanguineous family by WES. Western blotting and immunofluorescence experiments determined that the expression level of AK7 decreased in the sperm from the proband. The proband and his wife underwent two cycles of intracytoplasmic sperm injection (ICSI) treatment but got unfavorable outcomes. CONCLUSION: This study could provide precise genetic diagnosis for the patient and expand the spectrum of AK7 mutations.

The Effect of Heterozygous Mutation of Adenylate Kinase 2 Gene on Neutrophil Differentiation.

Mitochondrial ATP production plays an important role in most cellular activities, including growth and differentiation. Previously we reported that Adenylate kinase 2 (AK2) is the main ADP supplier in the mitochondrial intermembrane space in hematopoietic cells, especially in the bone marrow. AK2 is crucial for the production of neutrophils and T cells, and its deficiency causes reticular dysgenesis. However, the relationship between ADP supply by AK2 and neutrophil differentiation remains unclear. In this study, we used CRISPR/Cas9 technology to establish two heterozygous AK2 knock-out HL-60 clones as models for reticular dysgenesis. Their AK2 activities were about half that in the wild-type (WT). Furthermore, neutrophil differentiation was impaired in one of the clones. In silico analysis predicted that the obtained mutations might cause a structural change in AK2. Time course microarray analysis of the WT and mutants revealed that similar gene clusters responded to all-trans retinoic acid treatment, but their expression was lower in the mutants than in WT. Application of fructose partially restored neutrophil differentiation in the heterozygous knock-out HL-60 clone after all-trans retinoic acid treatment. Collectively, our study suggests that the mutation of N-terminal region in AK2 might play a role in AK2-dependent neutrophil differentiation and fructose could be used to treat AK2 deficiency.

Conditional disruption of AMP kinase in dopaminergic neurons promotes Parkinson's disease-associated phenotypes in vivo.

Emerging studies implicate energy dysregulation as an underlying trigger for Parkinson's disease (PD), suggesting that a better understanding of the molecular pathways governing energy homeostasis could help elucidate therapeutic targets for the disease. A critical cellular energy regulator is AMP kinase (AMPK), which we have previously shown to be protective in PD models. However, precisely how AMPK function impacts on dopaminergic neuronal survival and disease pathogenesis remains elusive. Here, we showed that Drosophila deficient in AMPK function exhibits PD-like features, including dopaminergic neuronal loss and climbing impairment that progress with age. We also created a tissue-specific AMPK-knockout mouse model where the catalytic subunits of AMPK are ablated in nigral dopaminergic neurons. Using this model, we demonstrated that loss of AMPK function promotes dopaminergic neurodegeneration and associated locomotor aberrations. Accompanying this is an apparent reduction in the number of mitochondria in the surviving AMPK-deficient nigral dopaminergic neurons, suggesting that an impairment in mitochondrial biogenesis may underlie the observed PD-associated phenotypes. Importantly, the loss of AMPK function enhances the susceptibility of nigral dopaminergic neurons in these mice to 6-hydroxydopamine-induced toxicity. Notably, we also found that AMPK activation is reduced in post-mortem PD brain samples. Taken together, these findings highlight the importance of neuronal energy homeostasis by AMPK in PD and position AMPK pathway as an attractive target for future therapeutic exploitation.

hCINAP is potentially a direct target gene of HIF-1 and is required for hypoxia-induced EMT and apoptosis in cervical cancer cells.

The early metastasis of cervical cancer is a multistep process requiring the cancer cells to adapt to the signal input from different tissue environments, including hypoxia. Hypoxia-induced epithelial-to-mesenchymal transition (EMT) plays a critical role in the ability to invade surrounding tissues. However, the molecular mechanisms underlying EMT in cervical cancer remain to be elucidated. Herein, we show that hypoxia-inducible factor-1alpha (HIF-1α) and aryl hydrocarbon receptor nuclear translocator (ARNT) are recruited to the human coilin-interacting nuclear ATPase protein (hCINAP) promoter and initiate hCINAP expression in hypoxia. Ablation of hCINAP decreased the migratory capacity and EMT of cervical cancer cells under hypoxic conditions. Furthermore, hCINAP regulated EMT through the Akt-mTOR signaling pathway, and inhibits hypoxia-induced p53-dependent apoptosis. Our data collectively show that hCINAP may have essential roles in the metastasis of cervical cancer and could be a potential target for curing cervical cancer.

Adenylate kinase AK2 isoform integral in embryo and adult heart homeostasis.

Adenylate kinase2 (AK2) catalyzes trans-compartmental nucleotide exchange, but the functional implications of this mitochondrial intermembrane isoform is only partially understood. Here, transgenic AK2-/- null homozygosity was lethal early in embryo, indicating a mandatory role for intact AK2 in utero development. In the adult, conditional organ-specific ablation of AK2 precipitated abrupt heart failure with Krebs cycle and glycolytic metabolite buildup, suggesting a vital contribution to energy demanding cardiac performance. Depressed pump function recovered to pre-deletion levels overtime, suggestive of an adaptive response. Compensatory upregulation of phosphotransferase AK1, AK3, AK4 isozymes, creatine kinase isoforms, and hexokinase, along with remodeling of cell cycle/growth genes and mitochondrial ultrastructure supported organ rescue. Taken together, the requirement of AK2 in early embryonic stages, and the immediate collapse of heart performance in the AK2-deficient postnatal state underscore a primordial function of the AK2 isoform. Unsalvageable in embryo, loss of AK2 in the adult heart was recoverable, underscoring an AK2-integrated bioenergetics system with innate plasticity to maintain homeostasis on demand.

Adenylate Kinase 4 Modulates the Resistance of Breast Cancer Cells to Tamoxifen through an m6A-Based Epitranscriptomic Mechanism.

N6-methyladenosine (m6A) is the most abundant internal modification in mRNA and this methylation constitutes an important regulatory mechanism for the stability and translational efficiency of mRNA. In this study, we found that the protein levels of adenylate kinase 4 (AK4) and m6A writer METTL3 are significantly higher in tamoxifen-resistant (TamR) MCF-7 cells than in parental cells. The TamR MCF-7 cells also exhibit increased methylation at multiple m6A consensus motif sites in the 5' untranslated region (5' UTR) of AK4 mRNA, and genetic depletion of METTL3 in TamR MCF-7 cells led to a diminished AK4 protein level and attenuated resistance to tamoxifen. In addition, we observed augmented levels of reactive oxygen species (ROS) and p38 activity in TamR MCF-7 cells, and both are diminished upon genetic depletion of AK4. Reciprocally, overexpression of AK4 in MCF-7 cells stimulates ROS and p38 phosphorylation levels, and it suppresses mitochondrial apoptosis. Moreover, scavenging of intracellular ROS leads to reduced p38 activity and re-sensitizes TamR MCF-7 cells to tamoxifen. Thus, our results uncover a novel m6A-mediated epitranscriptomic mechanism for the regulation of AK4, illustrate the cellular pathways through which increased AK4 expression contributes to tamoxifen resistance, and reveal AK4 as a potential therapeutic target for overcoming tamoxifen resistance.

Impacts of mutations on dynamic allostery of adenylate kinase.

Escherichia coli adenylate kinase (AK) is composed of CORE domain and two branch domains: LID and AMP-binding domain (AMPbd). AK exhibits considerable allostery in a reversible phosphoryl transfer reaction, which is largely attributed to the relative motion of LID and AMPbd with respect to CORE. Such an allosteric conformational change is also evident in the absence of ligands. Recent studies showed that the mutations in branch domains can adjust dynamic allostery and alter the substrate affinity and enzyme activity. In this work, we use all-atom molecular dynamics simulation to study the impacts of mutations in branch domains on AK's dynamic allostery by comparing two double mutants, i.e., LID mutant (Val135Gly, Val142Gly) and AMPbd mutant (Ala37Gly, Ala55Gly), with wild-type. Two mutants undergo considerable conformational fluctuation and exhibit deviation from the initially extended apo state to more compact structures. The LID domain in the LID mutant adjusts its relative position and firmly adheres to CORE. More strikingly, AMPbd mutations affect the relative positions of both the AMPbd domain and remote LID domain. Both domains undergo considerable movement, especially the inherent hinge swing motion of the flexible LID domain. In both mutants, the mutations can enhance the inter-domain interaction. The results about the conformation change of AK in both mutants are in line with the experiment of AK's affinity and activity. As revealed by our findings, the flexibility of branch domains and their inherent motions, especially LID domain, is highly relevant to dynamic allostery in the AK system.