Compensatory Interactions Research Articles

Two riboswitch classes that share a common ligand-binding fold show major differences in the ability to accommodate mutations.

Riboswitches are structured RNAs that sense small molecules to control expression. Prequeuosine1 (preQ1)-sensing riboswitches comprise three classes (I, II and III) that adopt distinct folds. Despite this difference, class II and III riboswitches each use 10 identical nucleotides to bind the preQ1 metabolite. Previous class II studies showed high sensitivity to binding-pocket mutations, which reduced preQ1 affinity and impaired function. Here, we introduced four equivalent mutations into a class III riboswitch, which maintained remarkably tight preQ1 binding.Co-crystal structures of each class III mutant showed compensatory interactions that preserve the fold. Chemical modification analysis revealed localized RNA flexibility changes for each mutant, but molecular dynamics (MD) simulations suggested that each mutation was not overtly destabilizing. Although impaired, class III mutants retained tangible gene-regulatory activity in bacteria compared to equivalent preQ1-II variants; mutations in the preQ1-pocket floor were tolerated better than wall mutations. Principal component analysis of MD trajectories suggested that the most functionally deleterious wall mutation samples different motions compared to wildtype. Overall, the results reveal that formation of compensatory interactions depends on the context of mutations within the overall fold and that functionally deleterious mutations can alter long-range correlated motions that link the riboswitch binding pocket with distal gene-regulatory sequences.

Expectancy-value interactions and dropout intentions in higher education: can study values compensate for low expectancies?

Research highlights the importance of expectancy-value interactions in predicting secondary-school students’ academic achievement. But as students transition to higher education, their expectancies and values undergo significant changes – highlighting the need to broaden the application of expectancy-value interactions to this context. To address this, we investigate the interactions between higher education students’ expectancies and their values in relation to students’ dropout intention. Data of N = 1140 students were analysed using latent moderated structural equation modelling. Similar to prior research, we find that expectancy-value interactions are related to students’ dropout intention. But rather than the synergistic interactions commonly found in the secondary-school context, we find that higher education students exhibit compensatory interactions: High study values and low costs could, to a certain degree, compensate for low expectancies. Furthermore, special attention should be paid to students who see little value in their studies and have a low success expectation, as they showed dropout intentions that far exceed effects indicated in prior research.

Compensatory Interactions between Corneal and Internal Astigmatism despite Lifestyle Changes.

This study explores whether children's refractive errors and visual behaviors reverted to pre-COVID-19 levels a year after normal schooling resumed in Hong Kong as well as the impact of corneal and internal astigmatism on refractive astigmatism development. Vision survey data and questionnaire results collected in 2022 (n = 119) and 2020 (n = 173) were compared. Cross-sectional data showed similar proportions of astigmatism (cylindrical power ≥ 0.75 D) in the 2020 (49.1%) and 2022 cohorts (55.5%). Despite a 0.28 D increase in corneal astigmatism, a compensatory 0.24 D increase in internal astigmatism of opposite direction kept refractive astigmatism relatively stable. The questionnaire data showed that children spent an additional 0.5 h/day outdoors on weekends post-resumption of normal schooling but engaged in more near-work activities, especially non-screen near-work, by approximately 1 h/day on both weekdays and weekends. These findings were supported by longitudinal data from 72 children who participated in both surveys. This study highlights the significant role of corneal and internal astigmatism in refractive astigmatism changes. Despite the return to in-person classes, children's total near-work time increased and astigmatism remained high. These findings underscore the need for comprehensive strategies to reduce the high environmental risks for refractive error development in children.

Modulation of Allostery with Multiple Mechanisms by Hotspot Mutations in TetR.

Modulating allosteric coupling offers unique opportunities for biomedical applications. Such efforts can benefit from efficient prediction and evaluation of allostery hotspot residues that dictate the degree of cooperativity between distant sites. We demonstrate that effects of allostery hotspot mutations can be evaluated qualitatively and semiquantitatively by molecular dynamics simulations in a bacterial tetracycline repressor (TetR). The simulations recapitulate the effects of these mutations on abolishing the induction function of TetR and provide a rationale for the different rescuabilities observed to restore allosteric coupling of the hotspot mutations. We demonstrate that the same noninducible phenotype could be the result of perturbations in distinct structural and energetic properties of TetR. Our work underscores the value of explicitly computing the functional free energy landscapes to effectively evaluate and rank hotspot mutations despite the prevalence of compensatory interactions and therefore provides quantitative guidance to allostery modulation for therapeutic and engineering applications.

Elevated carbon‐dioxide effects on wheat grain quality differed under contrasting nitrogen and phosphorus fertiliser supply

AbstractAtmospheric carbon‐dioxide concentration ([CO2]) is increasing rapidly, but its interactions with nitrogen (N) and phosphorus (P) fertiliser on wheat grain quality are not well understood. We investigated the effects of ambient CO2 (aCO2; ∼410 ppm) and elevated CO2 (eCO2; 760 ppm) on crop harvest index (CHI), nutrient harvest index (NuHI), shoot macro‐nutrient content and grain macro‐nutrient concentration of wheat grown under two contrasting amounts of N (0.5 and 6 mol m−3 NO3− N) and P (10 and 250 mmol P m−3) fertiliser supply (low and optimum, respectively). Our results highlighted interactions between [CO2] and N and P fertiliser supply for the shoot biomass at anthesis and straw biomass at harvest maturity. This was because biomass yield did not respond to CO2 level when fertiliser was deficient. However, shoot and straw yield increased (10.0–‐34.0%) with increasing [CO2] at optimum fertiliser rates. Across experiments, grain yield increased (15.6%) with increasing [CO2], which resulted in grain nutrient concentration decreasing (3.0–‐13.0%) with increasing [CO2]. This was attributed to nutrient 'dilution' due to increased carbohydrate content in the grain. Overall, fertiliser supply impacted crop responses more than CO2 treatments, and the impact was greater under N than P deficiency. This was reflected through conservative values for CHI, thousand grain weight and NuHIs suggesting plants allocated biomass and nutrients at similar rates for vegetative and reproductive organs independent of [CO2].

Direct-acting antiviral resistance of Hepatitis C virus is promoted by epistasis

Direct-acting antiviral agents (DAAs) provide efficacious therapeutic treatments for chronic Hepatitis C virus (HCV) infection. However, emergence of drug resistance mutations (DRMs) can greatly affect treatment outcomes and impede virological cure. While multiple DRMs have been observed for all currently used DAAs, the evolutionary determinants of such mutations are not currently well understood. Here, by considering DAAs targeting the nonstructural 3 (NS3) protein of HCV, we present results suggesting that epistasis plays an important role in the evolution of DRMs. Employing a sequence-based fitness landscape model whose predictions correlate highly with experimental data, we identify specific DRMs that are associated with strong epistatic interactions, and these are found to be enriched in multiple NS3-specific DAAs. Evolutionary modelling further supports that the identified DRMs involve compensatory mutational interactions that facilitate relatively easy escape from drug-induced selection pressures. Our results indicate that accounting for epistasis is important for designing future HCV NS3-targeting DAAs.

Neuron-oligodendrocyte potassium shuttling at nodes of Ranvier protects against inflammatory demyelination.

Multiple sclerosis (MS) is a progressive inflammatory demyelinating disease of the CNS. Increasing evidence suggests that vulnerable neurons in MS exhibit fatal metabolic exhaustion over time, a phenomenon hypothesized to be caused by chronic hyperexcitability. Axonal Kv7 (outward-rectifying) and oligodendroglial Kir4.1 (inward-rectifying) potassium channels have important roles in regulating neuronal excitability at and around the nodes of Ranvier. Here, we studied the spatial and functional relationship between neuronal Kv7 and oligodendroglial Kir4.1 channels and assessed the transcriptional and functional signatures of cortical and retinal projection neurons under physiological and inflammatory demyelinating conditions. We found that both channels became dysregulated in MS and experimental autoimmune encephalomyelitis (EAE), with Kir4.1 channels being chronically downregulated and Kv7 channel subunits being transiently upregulated during inflammatory demyelination. Further, we observed that pharmacological Kv7 channel opening with retigabine reduced neuronal hyperexcitability in human and EAE neurons, improved clinical EAE signs, and rescued neuronal pathology in oligodendrocyte-Kir4.1-deficient (OL-Kir4.1-deficient) mice. In summary, our findings indicate that neuron-OL compensatory interactions promoted resilience through Kv7 and Kir4.1 channels and identify pharmacological activation of nodal Kv7 channels as a neuroprotective strategy against inflammatory demyelination.

Chromatin complex dependencies reveal targeting opportunities in leukemia

Chromatin regulators are frequently mutated in human cancer and are attractive drug targets. They include diverse proteins that share functional domains and assemble into related multi-subunit complexes. To investigate functional relationships among these regulators, here we apply combinatorial CRISPR knockouts (KOs) to test over 35,000 gene-gene pairings in leukemia cells, using a library of over 300,000 constructs. Top pairs that demonstrate either compensatory non-lethal interactions or synergistic lethality enrich for paralogs and targets that occupy the same protein complex. The screen highlights protein complex dependencies not apparent in single KO screens, for example MCM histone exchange, the nucleosome remodeling and deacetylase (NuRD) complex, and HBO1 (KAT7) complex. We explore two approaches to NuRD complex inactivation. Paralog and non-paralog combinations of the KAT7 complex emerge as synergistic lethal and specifically nominate the ING5 PHD domain as a potential therapeutic target when paired with other KAT7 complex member losses. These findings highlight the power of combinatorial screening to provide mechanistic insight and identify therapeutic targets within redundant networks.

Genetic and Morphological Variation in Hypodontia of Maxillary Lateral Incisors.

(1) Background: Hypodontia has a multifactorial aetiology, in which genetic factors are a major component. Associated with this congenital absence, the formed teeth may show differences in size and shape, which may vary with the specific genetic variants and with the location of the missing teeth. The aims of the present study were to investigate a specific variant of MSX1, derive morphometric tooth measurements in a sample of patients with isolated maxillary lateral incisor agenesis and matched controls, and model the findings. (2) Methods: Genotyping of the MSX1 rs8670 genetic variant and morphometric measurements with a 2D image analysis method were performed for 26 hypodontia patients and 26 matched controls. (3) Results: The risk of upper lateral incisor agenesis was 6.9 times higher when the T allele was present. The morphometric parameters showed significant differences between hypodontia patients and controls and between the unilateral and bilateral agenesis cases. The most affected crown dimension in the hypodontia patients was the bucco-lingual dimension. In crown shape there was significant variation the Carabelli trait in upper first molars. (4) Conclusions: The MSX1 rs8670 variant was associated with variations in morphological outcomes. The new findings for compensatory interactions between the maxillary incisors indicate that epigenetic and environmental factors interact with this genetic variant. A single-level directional complex interactive network model incorporates the variations seen in this study.

A Hybrid SEM and Neural Network Approach to Understand and Predict the Determinants of Consumers’ Acceptance and Usage of Mobile-Commerce Application

The growth of mobile commerce marketplaces worldwide has been boosted by modern advances in digital technology. However, Privacy and security are still concern in m-commerce application. Since the previous study has researched the link between security and privacy and purpose to use, the factors that influence the formation of privacy and security in m-commerce are mostly unidentified. On the basis of UTAUT2, this study investigates the factors of security and privacy effecting mobile commerce acceptance. A hybrid SEM-ANN method was utilized to identify non-linear and compensatory interactions. Compensatory and Linear models are based on the idea that a deficiency in one component might also be compensated for by other variables. Linear and Non-compensatory models, on the other hand, seem to overcomplicate buyer decision mechanisms. Survey criteria have been conducted to obtain 890 mobile commerce consumer’s datasets utilizing an application on m-commerce. The following are the results. (1) M-commerce acceptability and use were positively influenced by five determinants (Security, performance expectancy, effort expectancy, habit, and price value). (2) Un-authorization, Error, secondary usage, collection, control, and awareness were all shown to directly and significantly negatively impact M-COMMERCE acceptance and use. (3) Three additional variables (social influence, hedonic motivation, and facilitating conditions) did not affect customers' intentions to use m-commerce applications in Jordan. In m-commerce, the integrated model expects a 45% percent increase in security and privacy.

Complex fitness landscape shapes variation in a hyperpolymorphic species.

It is natural to assume that patterns of genetic variation in hyperpolymorphic species can reveal large-scale properties of the fitness landscape that are hard to detect by studying species with ordinary levels of genetic variation. Here, we study such patterns in a fungus Schizophyllum commune, the most polymorphic species known. Throughout the genome, short-range linkage disequilibrium (LD) caused by attraction of minor alleles is higher between pairs of nonsynonymous than of synonymous variants. This effect is especially pronounced for pairs of sites that are located within the same gene, especially if a large fraction of the gene is covered by haploblocks, genome segments where the gene pool consists of two highly divergent haplotypes, which is a signature of balancing selection. Haploblocks are usually shorter than 1000 nucleotides, and collectively cover about 10% of the S. commune genome. LD tends to be substantially higher for pairs of nonsynonymous variants encoding amino acids that interact within the protein. There is a substantial correlation between LDs at the same pairs of nonsynonymous mutations in the USA and the Russian populations. These patterns indicate that selection in S. commune involves positive epistasis due to compensatory interactions between nonsynonymous alleles. When less polymorphic species are studied, analogous patterns can be detected only through interspecific comparisons.

Defining pseudoenzymes in glycosylation pathways

Glycosylation pathways regulate diverse cellular processes from protein quality control to host‐pathogen interactions and the enzymes that catalyze glycosidic linkages, glycosyltransferases (GTs), are a large super‐family of proteins that display extensive variation in primary sequence and three‐dimensional structure. A large fraction of GTs also display variations in key active site residues, but the functional and evolutionary implications of these variations are not well understood. In this talk, I will describe our ongoing efforts to map and classify GTs and pseudoGTs across the tree of life. A phylogenetic tree relating diverse GT‐A fold enzymes reveals that pseudoGTs evolved multiple independent times during evolution and variations in the catalytic base (xED‐Asp) correlate with the stereochemistry of glycosidic linkages in the acceptor substrate i.e., inverting vs retaining. Co‐evolutionary analysis reveals that active site variations in GTs are accommodated through compensatory interactions in distal functional sites associated with substrate recognition. Our studies support the “Red Queen” hypothesis in which the selective pressure to catalyze glycosidic linkages between diverse donor and acceptor substrates led to the emergence and expansion of pseudoGTs across the tree of life. Our findings have important implications for understanding other pseudoenzyme families such as pseudokinases.

Cognitive Rigidity, Habitual Tendencies, and Obsessive-Compulsive Symptoms: Individual Differences and Compensatory Interactions.

Recent theories have posited a range of cognitive risk factors for obsessive-compulsive disorder (OCD), including cognitive inflexibility and a maladaptive reliance on habits. However, empirical and methodological inconsistencies have obscured the understanding of whether inflexibility and habitual tendencies indeed shape OCD symptoms in clinical and sub-clinical populations, and whether there are notable interactions amongst these traits. The present investigation adopted an interactionist individual differences approach to examine the associations between behaviorally-assessed cognitive flexibility and subclinical OCD symptomatology in a healthy population. It also explored the nature of the interactions between cognitive flexibility and habitual tendencies, and the degree to which these cognitive traits predict subclinical OCD symptomatology. Across two studies, including a preregistration, Bayesian and regression analyses revealed that cognitive inflexibility and compulsive habitual tendencies act as unique and independent predictors of subclinical OCD symptomatology in healthy populations. Furthermore, there was a significant interaction between cognitive rigidity and habitual compulsivity, which accounted for 49.4% of the variance in subclinical OCD symptomatology in Study 1, and 37.3% in Study 2. In-depth analyses revealed a compensatory effect between cognitive inflexibility and habitual compulsivity such that both are necessary for OCD symptomatology, but neither is sufficient. These results imply that in order to generate reliable and nuanced models of the endophenotype of OCD symptomatology, it is essential to account for interactions between psychological traits. Moreover, the present findings have important implications for theories on the cognitive roots of OCD, and potentially in the development of interventions that target both cognitive inflexibility and habitual compulsivity.

Dopamine, Cognitive Flexibility, and IQ: Epistatic Catechol-O-MethylTransferase:DRD2 Gene-Gene Interactions Modulate Mental Rigidity.

Cognitive flexibility has been hypothesized to be neurochemically rooted in dopamine neurotransmission. Nonetheless, underpowered sample sizes and contradictory meta-analytic findings have obscured the role of dopamine genes in cognitive flexibility and neglected potential gene-gene interactions. In this largest neurocognitive-genetic study to date (n = 1400), single nucleotide polymorphisms associated with elevated prefrontal dopamine levels (catechol-O-methyltransferase; rs4680) and diminished striatal dopamine (C957T; rs6277) were both implicated in Wisconsin Card Sorting Test performance. Crucially, however, these genetic effects were only evident in low-IQ participants, suggesting high intelligence compensates for, and eliminates, the effect of dispositional dopamine functioning on flexibility. This interaction between cognitive systems may explain and resolve previous empirical inconsistencies in highly educated participant samples. Moreover, compensatory gene-gene interactions were discovered between catechol-O-methyltransferase and DRD2, such that genotypes conferring either elevated prefrontal dopamine or diminished striatal dopamine-via heightened striatally concentrated D2 dopamine receptor availability-are sufficient for cognitive flexibility, but neither is necessary. The study has therefore revealed a form of epistatic redundancy or substitutability among dopamine systems in shaping adaptable thought and action, thus defining boundary conditions for dopaminergic effects on flexible behavior. These results inform theories of clinical disorders and psychopharmacological interventions and uncover complex fronto-striatal synergies in human flexible cognition.

Do academic ability and social background influence each other in shaping educational attainment? The case of the transition to secondary education in Germany

Studies examining the interplay between children's academic ability and parental background are rare, and their findings are mixed and inconclusive. This paper addresses possible reinforcing or compensating relationships between child characteristics and parental social background in their influence on the transition to upper secondary education, the most important transition in the German school system. We use the German TwinLife data for genetically informed analyses and include comprehensive information on parental background as well as child cognitive ability and personality. We find no evidence for a significant influence of reinforcing or compensatory interactions in addition to the strong additive effects of child and parental characteristics. The ACE decomposition with covariates shows an almost equal influence of genetic variation and shared environments. However, indicators of child academic ability and parental background contribute only a little to explaining both contributions. Interestingly, the influence of child characteristics on enrollment in upper secondary school works mainly through environmental rather than genetic effects.

Evaluating interactions between emotion regulation strategies through the interpersonal context of female friends.

There is a growing interest in examining how interpersonal relationships may shape associations between emotion regulation (ER) strategies and psychopathology. We used multilevel modeling to test if respondents' self-reported intrapersonal ER, friends' self-reported intrapersonal ER, and their interaction were associated with psychopathology in a sample of 120 female friend dyads. Respondents' use of brooding rumination, expressive suppression, and worry were positively associated with respondent psychopathology. Friend reappraisal moderated the association between respondent reappraisal and respondent psychopathology. Consistent with an interference hypothesis, respondent cognitive reappraisal was only associated with respondent psychopathology when friend cognitive reappraisal was low. Consistent with a compensatory hypothesis, respondent reappraisal was primarily associated with respondent psychopathology when friend repetitive negative thought was high. Results support the extension of models of ER strategy interactions from intrapersonal to interpersonal contexts. Future research is needed to replicate the interference and compensatory interactions observed in the data.

Compensation of Adiponectin-Induced Adenosine Monophosphate-Activated Protein Kinase and p38 Mitogen-Activated Protein Kinase Signaling in Rheumatoid Arthritis Synovial Fibroblasts.

Rheumatoid arthritis (RA) is a chronic inflammatory disorder marked by synovitis, ultimately leading to cartilage and bone destruction. In RA, adiponectin levels are increased in serum and synovial fluid. Adiponectin belongs to the adipokines, a group of highly bioactive substances secreted by adipocytes and other cell types. It has been shown to induce the production of proinflammatory and prodestructive factors by human RA synovial fibroblasts (RASF), suggesting a role in the pathophysiology of the disease. Although adenosine monophosphate-activated protein kinase (AMPK) and p38 mitogen-activated protein kinase (MAPK) are known to be involved in adiponectin signaling in RASF, no literature is available about whether the different adiponectin isoforms affect AMPK and p38 MAPK signaling in the same manner. In this study, we elucidated the signaling mechanisms in RASF, activated in response to selective stimulation with the 2 biologically most potent adiponectin isoforms, and possible approaches to inhibit adiponectin-mediated effects in RASF. All adiponectin isoforms induced p38 MAPK and AMPK phosphorylation to various degrees. Blocking AMPK activation increased p38 MAPK phosphorylation, while blocking p38 MAPK activation increased AMPK phosphorylation, both independent of the effect of adiponectin. Neither AMPKα1 nor AMPKα2 knockdown reduced interleukin (IL)-6/IL-8 release. Targeting transforming growth factor-activated kinase 1 (TAK1), a signaling molecule upstream of p38 MAPK, reduced the IL-6/IL-8 release. Taken together, our study showed that, in the case of adiponectin isoforms, inhibiting the p38 MAPK or the AMPK signaling pathway individually is not sufficient, probably due to compensatory interactions between these pathways. TAK1 might provide an alternative approach by ameliorating the proinflammatory effects of adiponectin in RA. Our results do not suggest that targeting individual adiponectin isoforms specifically in RA would provide a benefit over targeting adiponectin as a whole. However, whether targeting individual adiponectin isoforms would allow minimizing the loss of the beneficial effects of adiponectin within the metabolic and cardiovascular system still needs further investigation.

Allele-specific suppression in Caenorhabditis elegans reveals details of EMS mutagenesis and a possible moonlighting interaction between the vesicular acetylcholine transporter and ERD2 receptors.

A missense mutant, unc-17(e245), which affects the Caenorhabditis elegans vesicular acetylcholine transporter UNC-17, has a severe uncoordinated phenotype, allowing efficient selection of dominant suppressors that revert this phenotype to wild-type. Such selections permitted isolation of numerous suppressors after EMS (ethyl methanesulfonate) mutagenesis, leading to demonstration of delays in mutation fixation after initial EMS treatment, as has been shown in T4 bacteriophage but not previously in eukaryotes. Three strong dominant extragenic suppressor loci have been defined, all of which act specifically on allele e245, which causes a G347R mutation in UNC-17. Two of the suppressors (sup-1 and sup-8/snb-1) have previously been shown to encode synaptic proteins able to interact directly with UNC-17. We found that the remaining suppressor, sup-2, corresponds to a mutation in erd-2.1, which encodes an endoplasmic reticulum retention protein; sup-2 causes a V186E missense mutation in transmembrane helix 7 of ERD-2.1. The same missense change introduced into the redundant paralogous gene erd-2.2 also suppressed unc-17(e245). Suppression presumably occurred by compensatory charge interactions between transmembrane helices of UNC-17 and ERD-2.1 or ERD-2.2, as previously proposed in work on suppression by SUP-1(G84E) or SUP-8(I97D)/synaptobrevin. erd-2.1(V186E) homozygotes were fully viable, but erd-2.1(V186E); erd-2.2(RNAi) exhibited synthetic lethality [like erd-2.1(RNAi); erd-2.2(RNAi)], indicating that the missense change in ERD-2.1 impairs its normal function in the secretory pathway but may allow it to adopt a novel moonlighting function as an unc-17 suppressor.

Native populations and the opioid crisis: forging a path to recovery

American Indian/Alaska Native (AI/AN) populations have proven particularly susceptible to the opioid crisis in the USA, but the White House’s 2019 national opioid policy roadmap is not structured to address AI/AN vulnerabilities. The concept of resilience, usually considered a positive system attribute, can be applied to complex systems to understand the larger compensatory interactions that restore systems to previous structures despite disruptions or interventions. The opioid crisis is a case of detrimental resilience because even effective interventions have not succeeded in eradicating opioid abuses. Resilience-based systemic interventions are needed to disrupt various aspects of systems while enhancing the social and cognitive abilities of affected populations to withstand the threat. This paper examines community characteristics, healthcare, and law enforcement within the context of AI/AN populations to emphasize the mechanisms that promote undesirable resilience for the opioid crisis. A research agenda bringing together systems science and management is needed to coordinate sectoral interventions and establish strategies to disrupt the resilient cycle of opioid addiction.

Effect of variations in the conserved residues E371 and S359 on the structural dynamics of protein Z dependent protease inhibitor (ZPI): a molecular dynamic simulation study

Protein Z (PZ) dependent protease inhibitor (ZPI) is a natural anticoagulant inhibiting blood coagulation proteases fXa and fXIa. Despite being a member of the serpin superfamily, it possesses unique structural features such as activation by PZ, regulating its inhibitory function. In order to understand the Reactive Centre Loop (RCL) dynamics of ZPI, which is absolutely critical for its activity, we performed Molecular Dynamics (MD) simulation on ZPI and its E371 and S359 variants located at important conserved functional sites. Unexpectedly, the RCL of E371 variants, (E371K, E371R, and E371Q), were shown to be very stable due to compensatory interactions at the proximal end of RCL. Interestingly, RCL flexibility was shown to be enhanced in the double mutant K318E-E371K due to the repulsive effect of increased negative charge on top of the breach region. Principal component analysis (PCA) coupled with residue wise interaction network analysis(RIN) revealed correlated motion between the RCL and the PZ binding regions in the WT. However, a loss of regulation in correlated motion between RCL and PZ binding hotspot Tyr240 in the double mutant was also observed. Additionally, the S359F and S359I mutations resulted in increased RCL flexibility owing to the disruption of stabilizing hydrogen bonding interaction at the distal end of strand S5A. Thus, the current study proposes that the overall stabilizing interactions of S5A is a major regulator of proper loop movement of ZPI for its activity. The results would be beneficial to engineer activity compromised ZPI as a prophylactic agent for the treatment of hemophilia. Communicated by Ramaswamy H. Sarma