Current Research in Structural Biology最新文献

{"title":"Molecular forces driving protein complexation of lentil and whey proteins: Structure-function relationships of trehalose-conjugated protein complexes on protein digestibility and solubility","authors":"Mohammad Alrosan ,&nbsp;Ali Madi Almajwal ,&nbsp;Ali Al-Qaisi ,&nbsp;Sana Gammoh ,&nbsp;Muhammad H. Alu'datt ,&nbsp;Farah R. Al Qudsi ,&nbsp;Thuan-Chew Tan ,&nbsp;Ammar A. Razzak Mahmood ,&nbsp;Sofyan Maghaydah","doi":"10.1016/j.crstbi.2024.100135","DOIUrl":"https://doi.org/10.1016/j.crstbi.2024.100135","url":null,"abstract":"<div><p>Plant-based proteins are often associated with a range of health benefits. Most research primarily investigates pea and soy proteins, while lentil proteins received minimal attention. This study evaluates the effect of protein complexation (using the pH-shifting technique) coupled with trehalose conjugation on lentil and whey proteins. The protein structures after the modification were analysed using spectroscopic methods: Fourier-transform infrared, ultraviolet spectra, and fluorescence spectra. The amide group I, conformation protein, and tertiary structure of the trehalose-conjugated lentil-whey protein complexes (T-LWPs) showed significant changes (<em>P &lt;</em> 0.05). Moreover, the surface properties (surface hydrophobicity and charges) of T-LWPs were significantly modified (<em>P &lt;</em> 0.05), from 457 to 324 a.u and from 36 to −40 mV, respectively. Due to these modifications on the protein structures, the protein digestibility (80–86%) and water solubility (90–94.5%) of T-LWPs increased significantly (<em>P</em> &lt; 0.05) with the increase in the trehalose concentration, from 0 (control) to 5% (w/w), respectively. This study suggested that coupling protein complexation and trehalose conjugation can enhance the overall properties of lentil-based protein complexes. With this enhancement, more opportunities in the utilisation of lentils are to be expected.</p></div>","PeriodicalId":10870,"journal":{"name":"Current Research in Structural Biology","volume":"7 ","pages":"Article 100135"},"PeriodicalIF":2.8,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2665928X24000126/pdfft?md5=5f24957755903c9c4bf2e15e12650a4f&pid=1-s2.0-S2665928X24000126-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140135101","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Back in time to the Gly-rich prototype of the phosphate binding elementary function","authors":"Zejun Zheng ,&nbsp;Alexander Goncearenco ,&nbsp;Igor N. Berezovsky","doi":"10.1016/j.crstbi.2024.100142","DOIUrl":"https://doi.org/10.1016/j.crstbi.2024.100142","url":null,"abstract":"<div><p>Binding of nucleotides and their derivatives is one of the most ancient elementary functions dating back to the Origin of Life. We review here the works considering one of the key elements in binding of (di)nucleotide-containing ligands – phosphate binding. We start from a brief discussion of major participants, conditions, and events in prebiotic evolution that resulted in the Origin of Life. Tracing back to the basic functions, including metal and phosphate binding, and, potentially, formation of primitive protein-protein interactions, we focus here on the phosphate binding. Critically assessing works on the structural, functional, and evolutionary aspects of phosphate binding, we perform a simple computational experiment reconstructing its most ancient and generic sequence prototype. The profiles of the phosphate binding signatures have been derived in form of position-specific scoring matrices (PSSMs), their peculiarities depending on the type of the ligands have been analyzed, and evolutionary connections between them have been delineated. Then, the apparent prototype that gave rise to all relevant phosphate-binding signatures had also been reconstructed. We show that two major signatures of the phosphate binding that discriminate between the binding of dinucleotide- and nucleotide-containing ligands are GxGxxG and GxxGxG, respectively. It appears that the signature archetypal for dinucleotide-containing ligands is more generic, and it can frequently bind phosphate groups in nucleotide-containing ligands as well. The reconstructed prototype's key signature GxGGxG underlies the role of glycine residues in providing flexibility and interactions necessary for binding the phosphate groups. The prototype also contains other ancient amino acids, valine, and alanine, showing versatility towards evolutionary design and functional diversification.</p></div>","PeriodicalId":10870,"journal":{"name":"Current Research in Structural Biology","volume":"7 ","pages":"Article 100142"},"PeriodicalIF":2.8,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2665928X24000199/pdfft?md5=3f93caa1ca09a9390f663f5d40a561d9&pid=1-s2.0-S2665928X24000199-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140555717","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"ANN based prediction of ligand binding sites outside deep cavities to facilitate drug designing","authors":"Kalpana Singh,&nbsp;Yashpal Singh Malik","doi":"10.1016/j.crstbi.2024.100144","DOIUrl":"https://doi.org/10.1016/j.crstbi.2024.100144","url":null,"abstract":"<div><p>The ever-changing environmental conditions and pollution are the prime reasons for the onset of several emerging and re-merging diseases. This demands the faster designing of new drugs to curb the deadly diseases in less waiting time to cure the animals and humans. Drug molecules interact with only protein surface on specific locations termed as ligand binding sites (LBS). Therefore, the knowledge of LBS is required for rational drug designing. Existing geometrical LBS prediction methods rely on search of cavities based on the fact that 83% of the LBS found in deep cavities, however, these methods usually fail where LBS localize outside deep cavities. To overcome this challenge, the present work provides an artificial neural network (ANN) based method to predict LBS outside deep cavities in animal proteins including human to facilitate drug designing. In the present work a feed-forward backpropagation neural network was trained by utilizing 38 structural, atomic, physiochemical, and evolutionary discriminant features of LBS and non-LBS residues localized in the extracted roughest patch on protein surface. The performance of this ANN based prediction method was found 76% better for those proteins where cavity subspace (extracted by MetaPocket 2.0, a consensus method) failed to predict LBS due to their localization outside the deep cavities. The prediction of LBS outside deep cavities will facilitate in drug designing for the proteins where it is not possible due to lack of LBS information as the geometrical LBS prediction methods rely on extraction of deep cavities.</p></div>","PeriodicalId":10870,"journal":{"name":"Current Research in Structural Biology","volume":"7 ","pages":"Article 100144"},"PeriodicalIF":2.8,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2665928X24000217/pdfft?md5=6488d8469a0b3dff9732a9f2ecb71a87&pid=1-s2.0-S2665928X24000217-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140606935","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Structural and functional insights of sortases and their interactions with antivirulence compounds","authors":"Sowmiya Sri Sivaramalingam ,&nbsp;Deepsikha Jothivel ,&nbsp;Deenadayalan Karaiyagowder Govindarajan ,&nbsp;Lohita Kadirvelu ,&nbsp;Muthusaravanan Sivaramakrishnan ,&nbsp;Dhivia Dharshika Chithiraiselvan ,&nbsp;Kumaravel Kandaswamy","doi":"10.1016/j.crstbi.2024.100152","DOIUrl":"10.1016/j.crstbi.2024.100152","url":null,"abstract":"<div><p>Sortase proteins play a crucial role as integral membrane proteins in anchoring bacterial surface proteins by recognizing them through a Cell-Wall Sorting (CWS) motif and cleaving them at specific sites before initiating pilus assembly. Both sortases and their substrate proteins are major virulence factors in numerous Gram-positive pathogens, making them attractive targets for antimicrobial intervention. Recognizing the significance of virulence proteins, a comprehensive exploration of their structural and functional characteristics is essential to enhance our understanding of pilus assembly in diverse Gram-positive bacteria. Therefore, this review article discusses the structural features of different classes of sortases and pilin proteins, primarily serving as substrates for sortase-assembled pili. Moreover, it thoroughly examines the molecular-level interactions between sortases and their inhibitors, providing insights from both structural and functional perspectives. In essence, this review article will provide a contemporary and complete understanding of both sortase pathways and various strategies to target them effectively to counteract the virulence.</p></div>","PeriodicalId":10870,"journal":{"name":"Current Research in Structural Biology","volume":"8 ","pages":"Article 100152"},"PeriodicalIF":2.8,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2665928X24000291/pdfft?md5=db64ca05515c00e67922cda0b3897a38&pid=1-s2.0-S2665928X24000291-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141395433","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The impact of SARS-CoV-2 spike mutation on peptide presentation is HLA allomorph-specific","authors":"You Min Ahn ,&nbsp;Janesha C. Maddumage ,&nbsp;Emma J. Grant ,&nbsp;Demetra S.M. Chatzileontiadou ,&nbsp;W.W.J. Gihan Perera ,&nbsp;Brian M. Baker ,&nbsp;Christopher Szeto ,&nbsp;Stephanie Gras","doi":"10.1016/j.crstbi.2024.100148","DOIUrl":"https://doi.org/10.1016/j.crstbi.2024.100148","url":null,"abstract":"<div><p>CD8⁺ T cells are crucial for viral elimination and recovery from viral infection. Nonetheless, the current understanding of the T cell response to SARS-CoV-2 at the antigen level remains limited. The Spike protein is an external structural protein that is prone to mutations, threatening the efficacy of current vaccines. Therefore, we have characterised the immune response towards the immunogenic Spike-derived peptide (S_976-984, VLNDILSRL), restricted to the HLA-A*02:01 molecule, which is mutated in both Alpha (S982A) and Omicron BA.1 (L981F) variants of concern. We determined that the mutation in the Alpha variant (S982A) impacted both the stability and conformation of the peptide, bound to HLA-A*02:01, in comparison to the original S_976-984. We identified a longer and overlapping immunogenic peptide (S_975-984, SVLNDILSRL) that could be presented by HLA-A*02:01, HLA-A*11:01 and HLA-B*13:01 allomorphs. We showed that S975-specific CD8⁺ T cells were weakly cross-reactive to the mutant peptides despite their similar conformations when presented by HLA-A*11:01. Altogether, our results show that the impact of SARS-CoV-2 mutations on peptide presentation is HLA allomorph-specific, and that post vaccination there are T cells able to react and cross-react towards the variant of concern peptides.</p></div>","PeriodicalId":10870,"journal":{"name":"Current Research in Structural Biology","volume":"7 ","pages":"Article 100148"},"PeriodicalIF":2.8,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2665928X24000254/pdfft?md5=68e3219bc99c70680b9c5efa9ff8b0ce&pid=1-s2.0-S2665928X24000254-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140824840","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Membrane binding and lipid-protein interaction of the C2 domain from coagulation factor V","authors":"Y. Zenmei Ohkubo ,&nbsp;Peter W. Radulovic ,&nbsp;Albert N. Kahira ,&nbsp;Jesper J. Madsen","doi":"10.1016/j.crstbi.2024.100149","DOIUrl":"https://doi.org/10.1016/j.crstbi.2024.100149","url":null,"abstract":"<div><p>Anchoring of coagulation factors to anionic regions of the membrane involves the C2 domain as a key player. The rate of enzymatic reactions of the coagulation factors is increased by several orders of magnitude upon membrane binding. However, the precise mechanisms behind the rate acceleration remain unclear, primarily because of a lack of understanding of the conformational dynamics of the C2-containing factors and corresponding complexes. We elucidate the membrane-bound form of the C2 domain from human coagulation factor V (FV–C2) by characterizing its membrane binding the specific lipid-protein interactions. Employing all-atom molecular dynamics simulations and leveraging the highly mobile membrane-mimetic (HMMM) model, we observed spontaneous binding of FV-C2 to a phosphatidylserine (PS)-containing membrane within 2–25 ns across twelve independent simulations. FV-C2 interacted with the membrane through three loops (spikes 1–3), achieving a converged, stable orientation. Multiple HMMM trajectories of the spontaneous membrane binding provided extensive sampling and ample data to examine the membrane-induced effects on the conformational dynamics of C2 as well as specific lipid-protein interactions. Despite existing crystal structures representing presumed “open” and “closed” states of FV-C2, our results revealed a continuous distribution of structures between these states, with the most populated structures differing from both “open” and “closed” states observed in crystal environments. Lastly, we characterized a putative PS-specific binding site formed by K23, Q48, and S78 located in the groove enclosed by spikes 1–3 (PS-specificity pocket), suggesting a different orientation of a bound headgroup moiety compared to previous proposals based upon analysis of static crystal structures.</p></div>","PeriodicalId":10870,"journal":{"name":"Current Research in Structural Biology","volume":"7 ","pages":"Article 100149"},"PeriodicalIF":2.8,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2665928X24000266/pdfft?md5=e8ab6e6a13338f75043488820ca37b17&pid=1-s2.0-S2665928X24000266-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140879519","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Cholesterol modulates the structural dynamics of the paddle motif loop of KvAP voltage sensor","authors":"Anindita Das ,&nbsp;Arpan Bysack ,&nbsp;H. Raghuraman","doi":"10.1016/j.crstbi.2024.100137","DOIUrl":"https://doi.org/10.1016/j.crstbi.2024.100137","url":null,"abstract":"<div><p>KvAP is a prokaryotic Kv channel, which has been widely used as a model system to understand voltage- and lipid-dependent gating mechanisms. In phospholipid membranes, the KvAP-VSD adopts the activated/‘Up’ conformation, whereas the presence of non-phospholipids in membranes favours the structural transition to resting/‘Down’ state. The S3b-S4 paddle motif loop of KvAP-VSD is functionally important as this participates in protein-protein interactions and is the target for animal toxins. In this study, we have monitored the modulatory role of cholesterol – the physiologically-relevant non-phospholipid – on the organization and dynamics of the S3b-S4 loop of the isolated KvAP-VSD in membranes by site-directed fluorescence approaches using the environmental sensitivity of 7-nitrobenz-2-oxa-1,3-diazol-4-yl-ethylenediamine (NBD) fluorescence. Our results show that cholesterol alters the dynamic nature (rotational and hydration dynamics) of S3b-S4 loop in a segmental fashion, <em>i.e</em>., the residues 110 to 114 and 115 to 117 behave differently in the presence of cholesterol, which is accompanied by considerable change in conformational heterogeneity. Further, quantitative depth measurements using the parallax quenching method reveal that the sensor loop is located at the shallow interfacial region of cholesterol-containing membranes, suggesting that the sensor loop organization is not directly correlated with S4 helix movement. Our results clearly show that cholesterol-induced changes in bilayer properties may not be the predominant factor for the sensor loop's altered structural dynamics, but can be attributed to the conformational change of the KvAP-VSD in cholesterol-containing membranes. Overall, these results are relevant for gating mechanisms, particularly the lipid-dependent gating, of Kv channels in membranes.</p></div>","PeriodicalId":10870,"journal":{"name":"Current Research in Structural Biology","volume":"7 ","pages":"Article 100137"},"PeriodicalIF":2.8,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2665928X2400014X/pdfft?md5=4e693bf08aef23c565c35af77c0c3c70&pid=1-s2.0-S2665928X2400014X-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140095959","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Structural insights into the initiation of free radical formation in the Class Ib ribonucleotide reductases in Mycobacteria","authors":"Lumbini R. Yadav ,&nbsp;Vasudha Sharma ,&nbsp;Maheswaran Shanmugam ,&nbsp;Shekhar C. Mande","doi":"10.1016/j.crstbi.2024.100157","DOIUrl":"10.1016/j.crstbi.2024.100157","url":null,"abstract":"<div><div>Class I ribonucleotide reductases consisting of α and β subunits convert ribonucleoside diphosphates to deoxyribonucleoside diphosphates involving an intricate free radical mechanism. The generation of free radicals in the Class Ib ribonucleotide reductases is mediated by di-manganese ions in the β subunits and is externally assisted by flavodoxin-like NrdI subunit. This is unlike Class Ia ribonucleotide reductases, where the free radical generation is initiated at its di-iron centre in the β subunits with no external support from another subunit. Class 1b ribonucleotide reductase complex is an essential enzyme complex in the human pathogen <em>Mycobacterium tuberculosis</em> and its structural details are largely unknown. In this study we have determined the crystal structures of Mycobacterial NrdI in oxidised and reduced forms, and similarly those of NrdF2:NrdI complexes. These structures provide detailed atomic view of the mechanism of free radical generation in the β subunit in this pathogen. We observe a well-formed channel in NrdI from the surface leading to the buried FMN moiety and propose that oxygen molecule accesses FMN through it. The oxygen molecule is further converted to a superoxide ion upon electron transfer at the FMN moiety. Similarly, a path for superoxide radical transfer between NrdI and NrdF2 is also observed. The oxidation of Mn(II) in NrdF2I to high valent oxidation state (either Mn(III) or Mn(IV) assisted by the reduced FMN site was evidently confirmed by EPR studies. SEC-MALS and low resolution cryo-EM map indicate unusual stoichiometry of 2:1 in the <em>M. tuberculosis</em> NrdF2I complex. A density close to Tyr 110 at a distance &lt;2.3 Å is observed, which we interpret as OH group. Overall, the study therefore provides important clues on the initiation of free radical generation in the β subunit of the ribonucleotide reductase complex in <em>M. tuberculosis</em>.</div></div>","PeriodicalId":10870,"journal":{"name":"Current Research in Structural Biology","volume":"8 ","pages":"Article 100157"},"PeriodicalIF":2.7,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142323032","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Conformational analysis of the IQSEC2 protein by statistical thermodynamics","authors":"Michael Shokhen ,&nbsp;Amnon Albeck ,&nbsp;Veronika Borisov ,&nbsp;Yonat Israel ,&nbsp;Nina S. Levy ,&nbsp;Andrew P. Levy","doi":"10.1016/j.crstbi.2024.100158","DOIUrl":"10.1016/j.crstbi.2024.100158","url":null,"abstract":"<div><div>Mutations in the IQSEC2 gene result in severe intellectual disability, epilepsy and autism. The primary function of IQSEC2 is to serve as a guanine exchange factor (GEF) controlling the activation of ARF6 which in turn mediates membrane trafficking and synaptic connections between neurons. As IQSEC2 is a large intrinsically disordered protein little is known of the structure of the protein and how this influences its function. Understanding this structure and function relationship is critical for the development of novel therapies to treat IQSEC2 disease. We therefore sought to identify IQSEC2 conformers in unfolded and folded states and analyze how conformers differ when binding to ARF6 and thereby influence GEF catalysis. We simulated the folding process of IQSEC2 by accelerated molecular dynamics (aMD). Following the ensemble method of Gibbs, we proposed that the number of microstates in the ensemble replicating a protein macroscopic system is the total number of MD snapshots sampled on the production MD trajectory. We divided the entire range of reaction coordinate into a series of consecutive, non-overlapping bins. Thermal fluctuations of biomolecules in local equilibrium states are Gaussian in form. To predict the free energy and entropy of different conformational states using statistical thermodynamics, the density of states was estimated taking into account how many MD snapshots constitute each conformational state. IQSEC2 dimers derived from the most stable folded and unfolded conformers of IQSEC2 were generated by protein-protein docking and then used to construct IQSEC2-ARF6 encounter complexes. We suggest that IQSEC2 folding and dimerization are two competing processes that may be used by nature to regulate the process of GDP exchange on ARF6 catalyzed by IQSEC2.</div></div>","PeriodicalId":10870,"journal":{"name":"Current Research in Structural Biology","volume":"8 ","pages":"Article 100158"},"PeriodicalIF":2.7,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142420737","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Stability and dynamics of extradenticle modulates its function","authors":"Aakanksha Singh,&nbsp;Bidisha Acharya,&nbsp;Beas Mukherjee,&nbsp;Veda Sheersh Boorla ,&nbsp;Soumendu Boral ,&nbsp;Snigdha Maiti ,&nbsp;Soumya De","doi":"10.1016/j.crstbi.2024.100150","DOIUrl":"https://doi.org/10.1016/j.crstbi.2024.100150","url":null,"abstract":"<div><p>Extradenticle (EXD) is a partner protein of the HOX transcription factors and plays an important role in the development of Drosophila. It confers increased affinity and specificity of DNA-binding to the HOX proteins. However, the DNA-binding homeodomain of EXD has a significantly weaker affinity to DNA compared to the HOX homeodomains. Here, we show that a glycine residue (G290) in the middle of the EXD DNA-binding helix primarily results in this weaker binding. Glycine destabilizes helices. To probe its role in the stability and function of the protein, G290 was mutated to alanine. The intrinsic stability of the DNA-binding helix increased in the G290A mutant as observed by NMR studies and molecular dynamics (MD) simulation. Also, NMR dynamics and MD simulation show that dynamic motions present in the wild-type protein are quenched in the mutant. This in turn resulted in increased stability of the entire homeodomain (ΔΔG_G→A of −2.6 kcal/mol). Increased protein stability resulted in three-fold better DNA-binding affinity of the mutant as compared to the wild-type protein. Molecular mechanics with generalized Born and surface area solvation (MMGBSA) analysis of our MD simulation on DNA-bound models of both wild-type and mutant proteins shows that the contribution to binding is enhanced for most of the interface residues in the mutant compared to the wild-type. Interestingly, the flexible N-terminal arm makes more stable contact with the DNA minor groove in the mutant. We found that the two interaction sites i.e. the DNA-binding helix and the unstructured N-terminal arm influence each other via the bound DNA. These results provide an interesting conundrum: alanine at position 290 enhances both the stability and the DNA-binding affinity of the protein, however, evolution prefers glycine at this position. We have provided several plausible explanations for this apparent conundrum. The function of the EXD as a HOX co-factor requires its ability to discriminate similar DNA sequences, which is most likely comprom</p></div>","PeriodicalId":10870,"journal":{"name":"Current Research in Structural Biology","volume":"7 ","pages":"Article 100150"},"PeriodicalIF":2.8,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2665928X24000278/pdfft?md5=fd7d76e526dba2e2fb02993ab8d46a86&pid=1-s2.0-S2665928X24000278-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140946903","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}