Interdomain Interactions Research Articles

Tackling APOE's structural challenges via in silico modeling in the era of neural networks: Can AlphaFold II help circumvent the problem of lacking full-length protein structure?

The APOE gene, encoding apolipoprotein E, is a predictor of longevity and age-related diseases. Despite numerous genetic studies, the data on molecular mechanisms by which apolipoprotein E affects the human phenotype remain incomplete due to the structural properties of the protein. Recently, a number of studies have used in silico drug discovery techniques based on protein-ligand docking, further highlighting the issue of lacking 3D structure of apolipoprotein E. Using molecular dynamics simulation, we found that AlphaFold II models of apolipoprotein E conformationally significantly differ both from the only available NMR structure, 2L7B, and structures obtained through circular dichroism spectroscopy: the ε4 isoform lacks the salt bridge between R61 and E255, while the ε2 and ε3 isoforms have extensive networks of interdomain interactions. Our findings challenge the benefits of using AlphaFold II for obtaining starting conformations for molecular docking.

Skin Bacterial and Fungal Microbiome Responses to Diet Supplementation and Rewilding in the Critically Endangered Southern Corroboree Frog.

The composition and dynamics of the skin bacterial and fungal microbiome is thought to influence host-pathogen defence. This microbial community is shaped by host captivity, diet, and microbial interactions between bacterial and fungal components. However, there remains little understanding of how specific micronutrients influence bacterial and fungal microbiome composition and their inter-domain interactions during rewilding of captive-bred animals. This study experimentally investigated the effect of dietary beta-carotene supplementation and subsequent field release on bacterial and fungal microbiome composition and dynamics using the Southern Corroboree frog (Pseudophryne corroboree) as a model system. We found large-scale diversification of bacterial communities post-release and similar diversification of fungal communities. The rewilded fungal mycobiome was more transient and demonstrated stronger temporal and micro-spatial fluctuations than the bacterial microbiome. Accounting for temporal and spatial factors, we found strong residual associations between bacterial members, yet limited evidence for inter-domain associations, suggesting that co-occurrence patterns between bacterial and fungal communities are largely a result of shared responses to the environment rather than direct interactions. Lastly, we found supplementation of dietary beta-carotene in captivity had no impact on post-release microbiome diversity, yet was associated with approximately 15% of common bacterial and fungal genera. Our research demonstrates that environmental factors play a dominant role over dietary beta-carotene supplementation in shaping microbiome diversity post-release, and suggest inter-domain interactions may also only exert a minor influence. Further research on the function and ecology of skin bacterial and fungal microbiomes will be crucial for developing strategies to support survival of endangered amphibian species.

Impact of N-Terminal Domain Conformation and Domain Interactions on RfaH Fold Switching.

RfaH is a two-domain metamorphic protein involved in transcription regulation and translation initiation. To carry out its dual functions, RfaH relies on two coupled structural changes: Domain dissociation and fold switching. In the free state, the C-terminal domain (CTD) of RfaH adopts an all-α fold and is tightly associated with the N-terminal domain (NTD). Upon binding to RNA polymerase (RNAP), the domains dissociate and the CTD transforms into an all-β fold while the NTD remains largely, but not entirely, unchanged. We test the idea that a change in the conformation of an extended β-hairpin (β3-β4) located on the NTD, helps trigger domain dissociation. To this end, we use homology modeling to construct a structure, H1, which is similar to free RfaH but with a remodeled β3-β4 hairpin. We then use an all-atom physics-based model enhanced with a dual basin structure-based potential to simulate domain separation driven by the thermal unfolding of the CTD with NTD in a fixed, folded conformation. We apply our model to both free RfaH and H1. For H1 we find, in line with our hypothesis, that the CTD exhibits lower stability and the domains dissociate at a lower temperature T, as compared to free RfaH. We do not, however, observe complete refolding to the all-β state in these simulations, suggesting that a change in β3-β4 orientation aids in, but is not sufficient for, domain dissociation. In addition, we study the reverse fold switch in which RfaH returns from a domain-open all-β state to its domain-closed all-α state. We observe a T-dependent transition rate; fold switching is slow at low T, where the CTD tends to be kinetically trapped in its all-β state, and at high-T, where the all-α state becomes unstable. Consequently, our simulations suggest an optimal T at which fold switching is most rapid. At this T, the stabilities of both folds are reduced. Overall, our study suggests that both inter-domain interactions and conformational changes within NTD may be important for the proper functioning of RfaH.

Domain Dynamics Response to Polarization Switching in Relaxor Ferroelectrics.

Nanoscale polar regions, or nanodomains (NDs), are crucial for understanding the domain structure and high susceptibility of relaxors. However, unveiling the evolution and function of NDs during polarization switching at the microscopic level is of great challenge. The experimental in situ characterization of NDs under electric-field perturbations, and computational accurate prediction of the dipole switching within a sufficiently large supercell, are notoriously tricky and tedious. These difficulties hinder a full understanding of the link between micro domain dynamics and macro polarization switching. Herein, the real-time evolution of NDs at the nanoscale is observed and visualized during polarization switching in an exemplary relaxor system of Bi5- xLaxMg0.5Ti3.5O15. Two fundamentally different domain switching pathways and dynamic characteristics are revealed: one steep, bipolar-like switching between two degenerate polarization states; and another flat, multi-step switching process with a thermodynamically stable non-polar mesophase mediating the degenerate polarization states. The two are determined by the distinct Landau energy landscapes that are strongly dependent on the intrinsic domain configurations and interdomain interactions. This work bridges the gap between micro domain dynamics and macro polarization switching, providing a guiding principle for the strategic design and optimization of relaxors.

Mechanistic Basis for a Single Amino Acid Residue Mutation Causing Human DNA Ligase 1 Deficiency, A Rare Pediatric Disease

In mammalian cells, DNA ligase 1 (LIG1) functions as the primary DNA ligase in both genomic replication and single-strand break repair. Several reported mutations in human LIG1, including R305Q, R641L, and R771W, cause LIG1 syndrome, a primary immunodeficiency. While the R641L and R771W mutations, respectively located in the nucleotidyl transferase and oligonucleotide binding domains, have been biochemically characterized and shown to reduce catalytic efficiency, the recently reported R305Q mutation within the DNA binding domain (DBD) remains mechanistically unexplored. The R641L and R771W mutations are known to decrease the catalytic activity of LIG1 by affecting both interdomain interactions and DNA binding during catalysis, without significantly impacting overall DNA affinity. To elucidate the molecular basis of the LIG1 syndrome-causing R305Q mutation, we purified this single-residue mutant protein and investigated its secondary structure, protein stability, DNA binding affinity, and catalytic efficiency. Our findings reveal that the R305Q mutation significantly impairs the function of LIG1 by disrupting the DBD-DNA interactions, leading to a 7–21-fold lower DNA binding affinity and a 33–300-fold reduced catalytic efficiency of LIG1. Additionally, the R305Q mutation slightly decreases LIG1’s protein stability by 2 to 3.6 °C, on par with the effect observed previously with either the R641L or R771W mutant. Collectively, our results uncover a new mechanism whereby the R305Q mutation impairs LIG1-catalyzed nicked DNA ligation, resulting in LIG1 syndrome, and highlight the crucial roles of the DBD-DNA interactions in tight DNA binding and efficient LIG1 catalysis.

Phosphorylation at the D56 residue of MtrA in Mycobacterium tuberculosis enhances its DNA binding affinity by modulating inter-domain interaction

The response regulator, MtrA, plays a major role in adaptation to the host environment, cell division, replication, and dormancy activation of Mycobacterium tuberculosis (Mtb). The phosphorylation of the response regulator MtrA alters the downstream activity, typically involving changes in DNA binding activity. However, there is a substantial knowledge gap in understanding the phosphorylation-mediated structural changes in MtrA. Additionally, the active conformation of the protein has yet to be determined. Therefore, in this study, we have investigated the phosphorylation-induced conformational changes of MtrA using all-atom molecular dynamics simulations under various phosphorylation conditions. The results from this study demonstrate that the phosphorylation at D56 (pD56-MtrA) increases the compactness of the MtrA protein by stabilizing the inter-domain interaction between the regulatory domain and DNA binding domain. Notably, the higher occupancy H-bond (over 95 %) between Arg200-Asn100 in case of the pD56-MtrA condition, which is otherwise absent in the non-phosphorylated (uMtrA) condition, suggests the importance of this interaction in the active conformation of the protein. The dynamic cross-correlation analysis reveals that phosphorylation (especially pD56-MtrA) reduces the anti-correlated motions and increases correlated motions between different domains. Moreover, the higher DNA binding affinity of pD56-MtrA compared to uMtrA supported by molecular docking and MD simulation followed by MMPBSA analysis suggests that pD56-MtrA is the possible active conformation of the MtrA protein. Overall, this investigation elucidates the key structural changes in MtrA under different phosphorylated conditions, which might help in designing novel therapeutics against tuberculosis.

Conformational ensembles in Klebsiella pneumoniae FimH impact uropathogenesis

Klebsiella pneumoniae is an important pathogen causing difficult-to-treat urinary tract infections (UTIs). Over 1.5 million women per year suffer from recurrent UTI, reducing quality of life and causing substantial morbidity and mortality, especially in the hospital setting. Uropathogenic E. coli (UPEC) is the most prevalent cause of UTI. Like UPEC, K. pneumoniae relies on type 1 pili, tipped with the mannose-binding adhesin FimH, to cause cystitis. However, K. pneumoniae FimH is a poor binder of mannose, despite a mannose-binding pocket identical to UPEC FimH. FimH is composed of two domains that are in an equilibrium between tense (low-affinity) and relaxed (high-affinity) conformations. Substantial interdomain interactions in the tense conformation yield a low-affinity, deformed mannose-binding pocket, while domain-domain interactions are broken in the relaxed state, resulting in a high-affinity binding pocket. Using crystallography, we identified the structural basis by which domain-domain interactions direct the conformational equilibrium of K. pneumoniae FimH, which is strongly shifted toward the low-affinity tense state. Removal of the pilin domain restores mannose binding to the lectin domain, thus showing that poor mannose binding by K. pneumoniae FimH is not an inherent feature of the mannose-binding pocket. Phylogenetic analyses of K. pneumoniae genomes found that FimH sequences are highly conserved. However, we surveyed a collection of K. pneumoniae isolates from patients with long-term indwelling catheters and identified isolates that possessed relaxed higher-binding FimH variants, which increased K. pneumoniae fitness in bladder infection models, suggesting that long-term residence within the urinary tract may select for higher-binding FimH variants.

Full-length structure and heme binding in the transcriptional regulator HcpR.

HcpR is a CRP-family transcriptional regulator found in many Gram-negative anaerobic bacteria. In the perio-pathogen Porphyromonas gingivalis, HcpR is crucial for the response to reactive nitrogen species such as nitric oxide (NO). Binding of NO to the heme group of HcpR leads to transcription of the redox enzyme Hcp. However, the molecular mechanisms of heme binding to HcpR remain unknown. In this study we present the 2.3Å structure of the P. gingivalis HcpR. Interdomain interactions present in the structure help to form a hydrophobic pocket in the N-terminal sensing domain. A comparison analysis with other CRP-family members reveals that the molecular mechanisms of HcpR-mediated regulation may be distinct from other family members. Using docking studies, we identify a putative heme binding site in the sensing domain. In vitro complementation and mutagenesis studies verify Met68 as an important residue in activation of HcpR. Finally, heme binding studies with purified forms of recombinant HcpR support Met68 and His149 residues as important for proper heme coordination in HcpR.

Identification of novel allosteric sites of SARS-CoV-2 papain-like protease (PLpro) for the development of COVID-19 antivirals

Coronaviruses such as SARS-CoV-2 encode a conserved papain-like protease (PLpro) that is crucial for viral replication and immune evasion, making it a prime target for antiviral drug development. In this study, three surface pockets on SARS-CoV-2 PLpro that may function as sites for allosteric inhibition were computationally identified. To evaluate the effects of these pockets on proteolytic activity, 52 residues were separately mutated to alanine. In Pocket 1, located between the Ubl and thumb domains, the introduction of alanine at T10, D12, T54, Y72, or Y83 reduced PLpro activity to <12% of that of WT. In Pocket 2, situated at the interface of the thumb, fingers, and palm domains, Q237A, S239A, H275A, and S278A inactivated PLpro. Finally, introducing alanine at five residues in Pocket 3, between the fingers and palm domains, inactivated PLpro: S212, Y213, Y251, K254, and Y305. Pocket 1 has a higher druggability score than Pockets 2 and 3. MD simulations showed that interactions within and between domains play critical roles in PLpro activity and thermal stability. The essential residues in Pockets 1 and 2 participate in a combination of intra- and inter-domain interactions. By contrast, the essential residues in Pocket 3 predominantly participate in inter-domain interactions. The most promising targets for therapeutic development are Pockets 1 and 3, which have the highest druggability score and the largest number of essential residues, respectively. Non-competitive inhibitors targeting these pockets may be antiviral agents against COVID-19 and related coronaviruses.

Hydrolysis Mechanism of Multimodular Endoglucanases with Distinctive Domain Composition in the Saccharification of Cellulosic Substrates.

Two multimodular endoglucanases in glycoside hydrolase family 5, ReCel5 and ElCel5, share 73% identity and exhibit similar modular structures: family 1 carbohydrate-binding module (CBM1); catalytic domain; CBMX2; module of unknown function. However, they differed in their biochemical properties and catalytic performance. ReCel5 showed optimal activity at pH 4.0 and 70 °C, maintaining stability at 70 °C (>80% activity). Conversely, ElCel5 is optimal at pH 3.0 and 50 °C (>50% activity at 50 °C). ElCel5 excels in degrading CMC-Na (256 U/mg vs 53 U/mg of ReCel5). Five domain-truncated (TM1-TM5) and four domain-replaced (RM1-RM4) mutants of ReCel5 with the counterparts of ElCel5 were constructed, and their enzymatic properties were compared with those of the wild type. Only RM1, with ElCel5-CBM1, displayed enhanced thermostability and activity. The hydrolysis of pretreated corn stover was reduced in most TM and RM mutants. Molecular dynamics simulations revealed interdomain interactions within the multimodular endoglucanase, potentially affecting its structural stability and complex biological catalytic processes.

Autoinhibition and relief mechanisms for MICAL monooxygenases in F-actin disassembly

MICAL proteins represent a unique family of actin regulators crucial for synapse development, membrane trafficking, and cytokinesis. Unlike classical actin regulators, MICALs catalyze the oxidation of specific residues within actin filaments to induce robust filament disassembly. The potent activity of MICALs requires tight control to prevent extensive damage to actin cytoskeleton. However, the molecular mechanism governing MICALs’ activity regulation remains elusive. Here, we report the cryo-EM structure of MICAL1 in the autoinhibited state, unveiling a head-to-tail interaction that allosterically blocks enzymatic activity. The structure also reveals the assembly of C-terminal domains via a tripartite interdomain interaction, stabilizing the inhibitory conformation of the RBD. Our structural, biochemical, and cellular analyses elucidate a multi-step mechanism to relieve MICAL1 autoinhibition in response to the dual-binding of two Rab effectors, revealing its intricate activity regulation mechanisms. Furthermore, our mutagenesis study of MICAL3 suggests the conserved autoinhibition and relief mechanisms among MICALs.

Revealing the principles of inter- and intra-domain regulation in a signaling enzyme via scanning mutagenesis.

Multi-domain enzymes can be regulated by both inter-domain interactions and structural features intrinsic to the catalytic domain. The tyrosine phosphatase SHP2 is a quintessential example of a multi-domain protein that is regulated by inter-domain interactions. This enzyme has a protein tyrosine phosphatase (PTP) domain and two phosphotyrosine-recognition domains (N-SH2 and C-SH2) that regulate phosphatase activity through autoinhibitory interactions. SHP2 is canonically activated by phosphoprotein binding to the SH2 domains, which causes large inter-domain rearrangements, but autoinhibition can also be disrupted by disease-associated mutations. Many details of the SHP2 activation mechanism are still unclear, the physiologically-relevant active conformations remain elusive, and hundreds of human variants of SHP2 have not been functionally characterized. Here, we perform deep mutational scanning on both full-length SHP2 and its isolated PTP domain to examine mutational effects on inter-domain regulation and catalytic activity. Our experiments provide a comprehensive map of SHP2 mutational sensitivity, both in the presence and absence of inter-domain regulation. Coupled with molecular dynamics simulations, our investigation reveals novel structural features that govern the stability of the autoinhibited and active states of SHP2. Our analysis also identifies key residues beyond the SHP2 active site that control PTP domain dynamics and intrinsic catalytic activity. This work expands our understanding of SHP2 regulation and provides new insights into SHP2 pathogenicity.

A conserved inhibitory interdomain interaction regulates DNA-binding activities of hybrid two-component systems in Bacteroides.

Different dietary and host-derived complex carbohydrates shape the gut microbial community and impact human health. In Bacteroides, the prevalent gut bacteria genus, utilization of these diverse carbohydrates relies on different gene clusters that are under sophisticated control by various signaling systems, including the hybrid two-component systems (HTCSs). We have uncovered a highly conserved regulatory mechanism in which the output DNA-binding activity of HTCSs is suppressed by interdomain interactions in the absence of stimulating phosphorylation. A consensus amino acid motif is found to correlate with the inhibitory interaction surface while deviations from the consensus can lead to constitutive activation. Understanding of such conserved HTCS features will be important to make regulatory predictions for individual systems as well as to engineer novel systems with substitutions in the consensus to explore the glycan regulation landscape in Bacteroides.

Halotolerant Bacillus sp. strain RA coordinates myo-inositol metabolism to confer salt tolerance to tomato.

Soil salinity is a worldwide problem threatening crop yields. Some plant growth-promoting rhizobacteria (PGPR) could survive in high salt environment and assist plant adaptation to stress. Nevertheless, the genomic and metabolic features, as well as the regulatory mechanisms promoting salt tolerance in plants by these bacteria remain largely unknown. In the current work, a novel halotolerant PGPR strain, namely, Bacillus sp. strain RA can enhance tomato tolerance to salt stress. Comparative genomic analysis of strain RA with its closely related species indicated a high level of evolutionary plasticity exhibited by strain-specific genes and evolutionary constraints driven by purifying selection, which facilitated its genomic adaptation to salt-affected soils. The transcriptome further showed that strain RA could tolerate salt stress by balancing energy metabolism via the reprogramming of biosynthetic pathways. Plants exude a plethora of metabolites that can strongly influence plant fitness. The accumulation of myo-inositol in leaves under salt stress was observed, leading to the promotion of plant growth triggered by Bacillus sp. strain RA. Importantly, myo-inositol serves as a selective force in the assembly of the phyllosphere microbiome and the recruitment of plant-beneficial species. It promotes destabilizing properties in phyllosphere bacterial co-occurrence networks, but not in fungal networks. Furthermore, interdomain interactions between bacteria and fungi were strengthened by myo-inositol in response to salt stress. This work highlights the genetic adaptation of RA to salt-affected soils and its ability to impact phyllosphere microorganisms through the adjustment of myo-inositol metabolites, thereby imparting enduring resistance against salt stress in tomato.

Mapping the Intersubunit Interdomain FMN-Heme Interactions in Neuronal Nitric Oxide Synthase by Targeted Quantitative Cross-Linking Mass Spectrometry.

Nitric oxide synthase (NOS) in mammals is a family of multidomain proteins in which interdomain electron transfer (IET) is controlled by domain-domain interactions. Calmodulin (CaM) binds to the canonical CaM-binding site in the linker region between the FMN and heme domains of NOS and allows tethered FMN domain motions, enabling an intersubunit FMN-heme IET in the output state for NO production. Our previous cross-linking mass spectrometric (XL MS) results demonstrated site-specific protein dynamics in the CaM-responsive regions of rat neuronal NOS (nNOS) reductase construct, a monomeric protein [Jiang et al., Biochemistry, 2023, 62, 2232-2237]. In this work, we have extended our combined approach of XL MS structural mapping and AlphaFold structural prediction to examine the homodimeric nNOS oxygenase/FMN (oxyFMN) construct, an established model of the NOS output state. We employed parallel reaction monitoring (PRM) based quantitative XL MS (qXL MS) to assess the CaM-induced changes in interdomain dynamics and interactions. Intersubunit cross-links were identified by mapping the cross-links onto top AlphaFold structural models, which was complemented by comparing their relative abundances in the cross-linked dimeric and monomeric bands. Furthermore, contrasting the CaM-free and CaM-bound nNOS samples shows that CaM enables the formation of the intersubunit FMN-heme docking complex and that CaM binding induces extensive, allosteric conformational changes across the NOS regions. Moreover, the observed cross-links sites specifically respond to changes in ionic strength. This indicates that interdomain salt bridges are responsible for stabilizing and orienting the output state for efficient FMN-heme IET. Taken together, our targeted qXL MS results have revealed that CaM and ionic strength modulate specific dynamic changes in the CaM/FMN/heme complexes, particularly in the context of intersubunit interdomain FMN-heme interactions.

Fungi play a crucial role in sustaining microbial networks and accelerating organic matter mineralization and humification during thermophilic phase of composting

Fungi play an important role in the mineralization and humification of refractory organic matter such as lignocellulose during composting. However, limited research on the ecological role of fungi in composting system hindered the development of efficient microbial agents. In this study, six groups of lab-scale composting experiments were conducted to reveal the role of fungal community in composting ecosystems by comparing them with bacterial community. The findings showed that the thermophilic phase was crucial for organic matter degradation and humic acid formation. The Richness index of the fungal community peaked at 1165 during this phase. PCoA analysis revealed a robust thermal stability in the fungal community. Despite temperature fluctuations, the community structure, predominantly governed by Pichia and Candida, remained largely unaltered. The stability of fungal community and the complexity of ecological networks were 1.26 times and 5.15 times higher than those observed in bacterial community, respectively. Fungi-bacteria interdomain interaction markedly enhanced network complexity, contributing to maintain microbial ecological functions. The core fungal species belonging to the family Saccharomycetaceae drove interdomain interaction during thermophilic phase. This study demonstrated the key role of fungi in the composting system, which would provide theoretical guidance for the development of high efficiency composting agents to strengthen the mineralization and humification of organic matter.

Significance of Zn2+ in RyR1 for Structural Integrity and Ligand Binding: Insight from Molecular Dynamics.

Ryanodine receptor type 1 (RyR1) is a Ca2+-release channel central to skeletal muscle excitation-contraction (EC) coupling. RyR1's cryo-EM structures reveal a zinc-finger motif positioned within the cytoplasmic C-terminal domain (CTD). Yet, owing to limitations in cryo-EM resolution, RyR1 structures lack precision in detailing the metal coordination structure, prompting the need for an accurate model. In this study, we employed molecular dynamics (MD) simulations and the density functional theory (DFT) method to refine the binding characteristics of Zn2+ in the zinc-finger site of the RyR1 channel. Our findings also highlight substantial conformational changes in simulations conducted in the absence of Zn2+. Notably, we observed a loss of contact at the interface between protein domains proximal to the zinc-finger site, indicating a crucial role of Zn2+ in maintaining structural integrity and interdomain interactions within RyR1. Furthermore, this study provides valuable insights into the modulation of ATP, Ca2+, and caffeine binding, shedding light on the intricate relationship between Zn2+ coordination and the dynamic behavior of RyR1. Our integrative approach combining MD simulations and DFT calculations enhances our understanding of the molecular mechanisms governing ligand binding in RyR1.

Dynamics of interdomain rotation facilitates FtsZ filament assembly

FtsZ, the tubulin homolog essential for bacterial cell division, assembles as Z-ring at the division site, and directs peptidoglycan synthesis by treadmilling. It is unclear how FtsZ achieves its kinetic polarity that drives treadmilling. To obtain insights into fundamental features of FtsZ assembly dynamics independent of peptidoglycan synthesis, we carried out the structural and biochemical characterization of FtsZ from the cell wall-less bacteria, Spiroplasma melliferum (SmFtsZ). Interestingly the structures of SmFtsZ, determined in GDP and GMPPNP bound form, were captured as domain swapped dimers. SmFtsZ was found to be a slower GTPase and has higher critical concentration (CC) for polymerization compared to Escherichia coli FtsZ (EcFtsZ). In FtsZs, a conformational switch from R-state (close) to T-state (open) favors polymerization. We identified a residue, Phe224, located at the interdomain cleft of SmFtsZ, which is crucial for R- to T-state transition. The mutation F224M in SmFtsZ cleft resulted in higher GTPase activity and lower CC, whereas the corresponding M225F in EcFtsZ resulted in cell division defects in E. coli. Our results demonstrate that relative rotation of the domains is a rate-limiting step of polymerization and that the dynamics of the interdomain rotation is important for the assembly of FtsZ filament. Our structural analysis of interdomain interactions suggests that this step is plausibly triggered upon addition of a GTP-bound monomer to the filament through interaction of the preformed N-terminal domain (NTD). Hence, the addition of monomers to the NTD-exposed end of filament is slower in comparison to the C-terminal domain (CTD) end, thus explaining kinetic polarity. In summary, the study sheds light on the molecular mechanisms underlying FtsZ assembly dynamics and highlights the importance of interdomain interactions, conformational changes, and specific residues in regulating FtsZ polymerization, which are crucial for bacterial cell division.

Contributions of the individual domains of αIIbβ3 integrin to its extension: Insights from multiscale modeling.

The platelet integrin αIIbβ3 undergoes long-range conformational transitions between bent and extended conformations to regulate platelet aggregation during hemostasis and thrombosis. However, how exactly αIIbβ3 transitions between conformations remains largely elusive. Here, we studied how transitions across bent and extended-closed conformations of αIIbβ3 integrin are regulated by effective interactions between its functional domains. We first carried out μs-long equilibrium molecular dynamics (MD) simulations of full-length αIIbβ3 integrins in bent and intermediate conformations, the latter characterized by an extended headpiece and closed legs. Then, we built heterogeneous elastic network models, perturbed inter-domain interactions, and evaluated their relative contributions to the energy barriers between conformations. Results showed that integrin extension emerges from: (i) changes in interfaces between functional domains; (ii) allosteric coupling of the head and upper leg domains with flexible lower leg domains. Collectively, these results provide new insights into integrin conformational activation based on short- and long-range interactions between its functional domains and highlight the importance of the lower legs in the regulation of integrin allostery.

Transient interdomain interactions in free USP14 shape its conformational ensemble.

The deubiquitinase (DUB) ubiquitin-specific protease 14 (USP14) is a dual domain protein that plays a regulatory role in proteasomal degradation and has been identified as a promising therapeutic target. USP14 comprises a conserved USP domain and a ubiquitin-like (Ubl) domain separated by a 25-residue linker. The enzyme activity of USP14 is autoinhibited in solution, but is enhanced when bound to the proteasome, where the Ubl and USP domains of USP14 bind to the Rpn1 and Rpt1/Rpt2 units, respectively. No structure of full-length USP14 in the absence of proteasome has yet been presented, however, earlier work has described how transient interactions between Ubl and USP domains in USP4 and USP7 regulate DUB activity. To better understand the roles of the Ubl and USP domains in USP14, we studied the Ubl domain alone and in full-length USP14 by nuclear magnetic resonance spectroscopy and used small angle x-ray scattering and molecular modeling to visualize the entire USP14 protein ensemble. Jointly, our results show how transient interdomain interactions between the Ubl and USP domains of USP14 predispose its conformational ensemble for proteasome binding, which may have functional implications for proteasome regulation and may be exploited in the design of future USP14 inhibitors.