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Approximately 3.4 million patients worldwide are diagnosed each year with cancers that have pathogenic mutations in one of three RAS proto-oncogenes (KRAS, NRAS and HRAS) 1,2 . These mutations impair the GTPase activity of RAS, leading to activation of downstream signalling and proliferation 3-6 . Long-standing efforts to restore the hydrolase activity of RAS mutants have been unsuccessful, extinguishing any consideration towards a viable therapeutic strategy 7 . Here we show that tri-complex inhibitors-that is, molecular glues with the ability to recruit cyclophilin A (CYPA) to the active state of RAS-have a dual mechanism of action: not only do they prevent activated RAS from binding to its effectors, but they also stimulate GTP hydrolysis. Drug-bound CYPA complexes modulate residues in the switch II motif of RAS to coordinate the nucleophilic attack on the γ-phosphate of GTP in a mutation-specific manner. RAS mutants that were most sensitive to stimulation of GTPase activity were more susceptible to treatment than mutants in which the hydrolysis could not be enhanced, suggesting that pharmacological stimulation of hydrolysis potentiates the therapeutic effects of tri-complex inhibitors for specific RAS mutants. This study lays the foundation for developing a class of therapeutics that inhibit cancer growth by stimulating mutant GTPase activity., Competing Interests: Competing interests: P.L. is listed as an inventor on patents filed by MSKCC regarding treatment of KRAS- or BRAF-mutant cancers; reports grants to his institution from Revolution Medicines, Amgen, Mirati and Boehringer Ingelheim; and reports consulting fees or honoraria from Black Diamond Therapeutics, AmMax, OrbiMed, PAQ-Tx, Repare Therapeutics, Boehringer Ingelheim, Menarini Group and Revolution Medicines, as well as membership on the scientific advisory board of Frontier Medicines, Ikena, Biotheryx and PAQ-Tx (consulting fees and equity in each). The other authors declare no competing interests., (© 2024. The Author(s).)

Proton transfer reactions play important functional roles in many proteins, such as enzymes and transporters, which is also the case in rhodopsins. In fact, functional expression of rhodopsins accompanies intramolecular proton transfer reactions in many cases. One of the exceptional cases can be seen in the protonated form of marine bacterial TAT rhodopsin, which isomerizes the retinal by light but returns to the original state within 10 -5 s. Thus, light energy is converted into heat without any function. In contrast, the T82D mutant of TAT rhodopsin conducts the light-induced deprotonation of the Schiff base at high pH. In this article, we report the structural analysis of T82D by means of difference Fourier transform infrared (FTIR) spectroscopy. In the light-induced difference FTIR spectra at 77 K, we observed little hydrogen out-of-plane vibrations for T82D as well as the wild-type (WT), suggesting that the planar chromophore structure itself is not the origin of the reversion from the K intermediate in WT TAT rhodopsin. Upon relaxation of the K intermediate, T82D forms the following intermediate, such as M, whereas K of WT returns to the original state. Present FTIR analysis revealed the proton transfer from the Schiff base to D82 in T82D upon formation of the M intermediate. It is accompanied by the second proton transfer from E54 to the Schiff base, forming the N intermediate, particularly in membranes. The equilibrium between the M and N intermediates corresponds to the protonation equilibrium between E54 and the Schiff base. We also found that Ca 2+ binding takes place in T82D as well as WT but with 6 times lower affinity. An altered hydrogen-bonding network would be the origin of low affinity in T82D, where deprotonation of E54 is involved in the Ca 2+ binding., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024. Published by Elsevier Inc.)

Protein-water interactions profoundly influence protein structure and dynamics. Consequently, the function of many biomacromolecules is directly related to the presence and exchange of water molecules. While structural water molecules can be readily identified through X-ray crystallography, the dynamics within functional protein-water networks remain largely elusive. Therefore, to understand the role of biological water in protein dynamics and function, we have introduced S2A and H64A mutations in human Carbonic Anhydrase II (hCAII), a model system to study protein-water interactions. The mutations of serine to alanine at position 2 and histidine to alanine at position 64 cause an increase in hydrophobicity in the N-terminus and active site loop thereby restricting water entry and disrupting the water network in the Zn 2+ -binding pocket. To pave the way for a detailed investigation into the structural, functional, and mechanistic aspects of the Ser2Ala/His64Ala double mutant of hCAII, we present here almost complete sequence-specific resonance assignments for 1 H, 15 N, and 13 C. These assignments serve as the basis for comprehensive studies on the dynamics of the protein-water network within the Zn 2+ -binding pocket and its role in catalysis., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

The potency of frontline antimalarial drug artemisinin (ART) derivatives is triggered by heme-induced cleavage of the endoperoxide bond to form reactive heme-ART alkoxy radicals and covalent heme-ART adducts, which are highly toxic to the parasite. ART-resistant (ART-R) parasites with mutations in the Plasmodium falciparum Kelch-containing protein Kelch13 (PfKekch13) exhibit impaired hemoglobin uptake, reduced yield of hemoglobin-derived heme, and thus decreased ART activation. However, any direct involvement of PfKelch13 in heme-mediated ART activation has not been reported. Here, we show that the purified recombinant PfKelch13 wild-type (WT) protein displays measurable binding affinity for iron and heme, the main effectors for ART activation. The heme-binding property is also exhibited by the native PfKelch13 protein from parasite culture. The two ART-R recombinant PfKelch13 mutants (C580Y and R539T) display weaker heme binding affinities compared to the ART-sensitive WT and A578S mutant proteins, which further translates into reduced yield of heme-ART derivatives when ART is incubated with the heme molecules bound to the mutant PfKelch13 proteins. In conclusion, this study provides the first evidence for ART activation via the heme-binding propensity of PfKelch13. This mechanism may contribute to the modulation of ART-R levels in malaria parasites through a novel function of PfKelch13., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

The Kirsten Rat Sarcoma (KRAS) G12D mutant protein is a primary driver of pancreatic ductal adenocarcinoma, necessitating the identification of targeted drug molecules. Repurposing of drugs quickly finds new uses, speeding treatment development. This study employs microsecond molecular dynamics simulations to unveil the binding mechanisms of the FDA-approved MEK inhibitor trametinib with KRAS G12D , providing insights for potential drug repurposing. The binding of trametinib was compared with clinical trial drug MRTX1133, which demonstrates exceptional activity against KRAS G12D , for better understanding of interaction mechanism of trametinib with KRAS G12D . The resulting stable MRTX1133-KRAS G12D complex reduces root mean square deviation (RMSD) values, in Switch I and II domains, highlighting its potential for inhibiting KRAS G12D . MRTX1133's robust interaction with Tyr64 and disruption of Tyr96-Tyr71-Arg68 network showcase its ability to mitigate the effects of the G12D mutation. In contrast, trametinib employs a distinctive binding mechanism involving P-loop, Switch I and II residues. Extended simulations to 1 μs reveal sustained network interactions with Tyr32, Thr58, and GDP, suggesting a role of trametinib in maintaining KRAS G12D in an inactive state and impede the further cell signaling. The decomposition binding free energy values illustrate amino acids' contributions to binding energy, elucidating ligand-protein interactions and molecular stability. The machine learning approach reveals that van der Waals interactions among the residues play vital role in complex stability and the potential amino acids involved in drug-receptor interactions of each complex. These details provide a molecular-level understanding of drug binding mechanisms, offering essential knowledge for further drug repurposing and potential drug discovery., (© 2024 John Wiley & Sons Ltd.)

Misfolding of the prion protein is linked to multiple neurodegenerative diseases. A better understanding of the process requires the identification and structural characterization of intermediate conformations via which misfolding proceeds. In this study, three conserved aromatic residues (Tyr168, Phe174, and Tyr217) located in the C-terminal domain of mouse PrP (wt moPrP) were mutated to Ala. The resultant mutant protein, 3A moPrP, is shown to adopt a molten globule (MG)-like native conformation. Hydrogen-deuterium exchange studies coupled with mass spectrometry revealed that for 3A moPrP, the free energy gap between the MG-like native conformation and misfolding-prone partially unfolded forms is reduced. Consequently, 3A moPrP misfolds in native conditions even in the absence of salt, unlike wt moPrP, which requires the addition of salt to misfold. 3A moPrP misfolds to a β-rich dimer in the absence of salt, which can rapidly form an oligomer upon the addition of salt. In the presence of salt, 3A moPrP misfolds to a β-rich oligomer about a thousand-fold faster than wt moPrP. Importantly, the misfolded structure of the dimer is similar to that of the salt-induced oligomer. Misfolding to oligomer seems to be induced at the level of the dimeric unit by monomer-monomer association, and the oligomer grows by accretion of misfolded dimeric units. Additionally, it is shown that the conserved aromatic residues collectively stabilize not only monomeric protein, but also the structural core of the β-rich oligomers. Finally, it is also shown that 3A moPrP misfolds much faster to amyloid-fibrils than does the wt protein., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Vitamin D (VD) is produced by the skin upon exposure to sunlight or is obtained from dietary sources. Several risk factors are associated with VD deficiency including mutations and post-translational modifications in its transport protein known as vitamin D binding protein (VDBP) or GC-globulin. The two common single nucleotide polymorphisms rs7041 and rs4588 create three major isoforms of VDBP, including GC-1F also called wild type, GC1S, and GC-2. The 3D models for both GC-1F and GC-2 were constructed in their glycosylated states to decipher the effect of these mutations on the overall conformational changes and VD-binding affinity. The binding affinities were estimated using the Molecular Mechanics Poison-Boltzmann surface area (MM-PBSA) method and conformational changes were investigated after free energy landscapes estimations. Total free energies suggest that GC-1F exhibits stronger affinity (ΔE = -116.09 kJ/mol) than GC-2 (ΔE = -95 kJ/mol) variant with VD. The GC-1F isoforms had more streamlined motion compared to GC-2 isoforms, predicting a trade-off between cross-talk residues that significantly impacts protein structural stability. The data suggest that glycation at Thr418 plays a vital role in the overall VDBP-VD affinity by stabilizing the N-T loop that holds the domain I (VD-pocket) and domain III intact. The loss of glycation in GC-2 has a pivotal role in the inter-domain conformational stability of VDBP, which may ultimately affect VD transportation and maturation. These findings describe a novel mechanism in how mutations distant from the VD-active site change the overall conformational of the VDBP and abrogate the VDBP-VD interaction.Communicated by Ramaswamy H. Sarma.

In preparation for a detailed exploration of the structural and functional aspects of the Ser2Ala mutant of human carbonic anhydrase II, we present here almost complete sequence-specific resonance assignments for 1 H, 15 N, and 13 C. The mutation of serine to alanine at position 2, located in the N-terminal region of the enzyme, significantly alters the hydrophilic nature of the site, rendering it hydrophobic. Consequently, there is an underlying assumption that this mutation would repel water from the site. However, intriguingly, comparative analysis of the mutant structure with the wild type reveals minimal discernible differences. These assignments serve as the basis for in-depth studies on histidine dynamics, protonation states, and its intricate role in protein-water interactions and catalysis., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Human K-Ras protein, which is a member of the GTPase Ras family, hydrolyzes GTP to GDP and concomitantly converts from its active to its inactive state. It is a key oncoprotein, because several mutations, particularly those at residue position 12, occur with a high frequency in a wide range of human cancers. The K-Ras protein is therefore an important target for developing therapeutic anti-cancer agents. In this work we report the almost complete sequence-specific resonance assignments of wild-type and the oncogenic G12C and G12D mutants in the GTP-complexed active forms, including the functionally important Switch I and Switch II regions. These assignments serve as the basis for a comprehensive functional dynamics study of wild-type K-Ras and its G12 mutants., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

Uric acid is the end product of purine metabolism in humans due to inactivation of the uricase determined by the mutated uricase gene. Uricase catalyzes the conversion of uric acid into water-soluble allantoin that is easily excreted by the kidneys. Hyperuricemia occurs when the serum concentration of uric acid exceeds its solubility (7 mg/dL). However, modifications to improve the uricase activity is under development for treating the hyperuricemia. Here we designed 7 types of human-porcine chimeric uricase by multiple sequence comparisons and targeted mutagenesis. An optimal human-porcine chimeric uricase mutant (uricase-10) with both high activity (6.33 U/mg) and high homology (91.45 %) was determined by enzyme activity measurement. The engineering uricase was further modified with PEGylation to improve the stability of recombinant protein drugs and reduce immunogenicity, uricase-10 could be more suitable for the treatment of gout and hyperuricemia theoretically., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

α-Synuclein, a presynaptic neuronal protein encoded by the SNCA gene, is involved in the pathogenesis of Parkinson's disease. Point mutations and multiplications of α-synuclein (A30P and A53T) are correlated with early-onset Parkinson's disease characterized by rapid progression and poor prognosis. Currently, the clinical identification of SNCA variants, especially disease-related A30P and A53T mutants, remains challenging and also time consuming. This study aimed to develop a novel label-free detection method for distinguishing the SNCA mutants using transmission terahertz (THz) time-domain spectroscopy. The protein was spin-coated onto the quartz to form a thin film, which was measured using THz time-domain spectroscopy. The spectral characteristics of THz broadband pulse waves of α-synuclein protein variants (SNCA wild type, A30P, and A53T) at different frequencies were analyzed via Fourier transform. The amplitude A intensity (A WT , A A30P , and A A53T ) and peak occurrence time in THz time-domain spectroscopy sensitively distinguished the three protein variants. The phase φ difference in THz frequency domain followed the trend of φ WT  > φ A30P  > φ A53T . There was a significant difference in THz frequency amplitude A' corresponding to the frequency ranging from 0.4 to 0.66 THz (A' A53T  > A' A30P  > A' WT ). At a frequency of 0.4-0.6 THz, the transmission T of THz waves distinguished three variants (T A53T  > T A30P  > T WT ), whereas there was no difference in the transmission T at 0.66 THz. The SNCA wild-type protein and two mutant variants (A30P and A53T) had distinct characteristic fingerprint spectra on THz time-domain spectroscopy. This novel label-free detection method has great potential for the differential diagnosis of Parkinson's disease subtypes., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

αB-crystallin, a member of the small heat shock protein (sHSP) family, is expressed in diverse tissues, including the eyes, brain, muscles, and heart. This protein plays a crucial role in maintaining eye lens transparency and exhibits holdase chaperone and anti-apoptotic activities. Therefore, structural and functional changes caused by genetic mutations in this protein may contribute to the development of disorders like cataract and cardiomyopathy. Recently, the substitution of arginine 123 with tryptophan (p.R123W mutation) in human αB-crystallin has been reported to trigger cardiomyopathy. In this study, human αB-crystallin was expressed in Escherichia coli (E. coli), and the missense mutation p.R123W was created using site-directed mutagenesis. Following purification via anion exchange chromatography, the structural and functional properties of both proteins were investigated and compared using a wide range of spectroscopic and microscopic methods. The p.R123W mutation induced significant alterations in the secondary, tertiary, and quaternary structures of human αB-crystallin. This pathogenic mutation resulted in an increased β-sheet structure and formation of protein oligomers with larger sizes compared to the wild-type protein. The mutant protein also exhibited reduced chaperone activity and lower thermal stability. Atomic force microscopy (AFM) and transmission electron microscopy (TEM) demonstrated that the p.R123W mutant protein is more prone to forming amyloid aggregates. The structural and functional changes observed in the p.R123W mutant protein, along with its increased propensity for aggregation, could impact its proper functional interaction with the target proteins in the cardiac muscle, such as calcineurin. Our results provide an explanation for the pathogenic intervention of p.R123W mutant protein in the occurrence of hypertrophic cardiomyopathy (HCM)., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Reza Yousefi reports financial support was provided by Iran National Science Foundation. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

αB-Crystallin (αB-Cry) is expressed in many tissues, and mutations in this protein are linked to various diseases, including cataracts, Alzheimer's disease, Parkinson's disease, and several types of myopathies and cardiomyopathies. The p.D109G mutation, which substitutes a conserved aspartate residue involved in the interchain salt bridges, with glycine leads to the development of both restrictive cardiomyopathy (RCM) and skeletal myopathy. In this study, we generated this mutation in the α-Cry domain (ACD) which is crucial for forming the active chaperone dimeric state, using site-directed mutagenesis. After inducing expression in the bacterial host, we purified the mutant and wild-type recombinant proteins using anion exchange chromatography. Various spectroscopic evaluations revealed significant changes in the secondary, tertiary, and quaternary structures of human αB-Cry caused by this mutation. Furthermore, this pathogenic mutation led to the formation of protein oligomers with larger sizes than those of the wild-type protein counterpart. The mutant protein also exhibited increased chaperone activity and decreased chemical, thermal, and proteolytic stability. Atomic force microscopy (AFM), transmission electron microscopy (TEM), and fluorescence microscopy (FM) demonstrated that p.D109G mutant protein is more prone to forming amyloid aggregates. The misfolding associated with the p.D109G mutation may result in abnormal interactions of human αB-Cry with its natural partners (e.g., desmin), leading to the formation of protein aggregates. These aggregates can interfere with normal cellular processes and may contribute to muscle cell dysfunction and damage, resulting in the pathogenic involvement of the p.D109G mutant protein in restrictive cardiomyopathy and skeletal myopathy., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The study of protein folding plays a crucial role in improving our understanding of protein function and of the relationship between genetics and phenotypes. In particular, understanding the thermodynamics and kinetics of the folding process is important for uncovering the mechanisms behind human disorders caused by protein misfolding. To address this issue, it is essential to collect and curate experimental kinetic and thermodynamic data on protein folding. K-Pro is a new database designed for collecting and storing experimental kinetic data on monomeric proteins, with a two-state folding mechanism. With 1,529 records from 62 proteins corresponding to 65 structures, K-Pro contains various kinetic parameters such as the logarithm of the folding and unfolding rates, Tanford's β and the ϕ values. When available, the database also includes thermodynamic parameters associated with the kinetic data. K-Pro features a user-friendly interface that allows browsing and downloading kinetic data of interest. The graphical interface provides a visual representation of the protein and mutants, and it is cross-linked to key databases such as PDB, UniProt, and PubMed. K-Pro is open and freely accessible through https://folding.biofold.org/k-pro and supports the latest versions of popular browsers., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Machine-learning algorithm uses structure prediction to spot disease-causing mutations.

Stability is critical for the proper functioning of all proteins. Optimization of protein thermostability is a key step in the development of industrial enzymes and biologics. Herein, we demonstrate that multidomain proteins can be stabilized significantly using domain-based engineering followed by the recombination of the optimized domains. Domain-level analysis of designed protein variants with similar structures but different thermal profiles showed that the independent enhancement of the thermostability of a constituent domain improves the overall stability of the whole multidomain protein. The crystal structure and AlphaFold-predicted model of the designed proteins via domain-recombination provided a molecular explanation for domain-based stepwise stabilization. Our study suggests that domain-based modular engineering can minimize the sequence space for calculations in computational design and experimental errors, thereby offering useful guidance for multidomain protein engineering., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Congenital myasthenic syndrome (CMS) is a heterogeneous condition associated with 34 different genes, including SLC5A7, which encodes the high-affinity choline transporter 1 (CHT1). CHT1 is expressed in presynaptic neurons of the neuromuscular junction where it uses the inward sodium gradient to reuptake choline. Biallelic CHT1 mutations often lead to neonatal lethality, and less commonly to non-lethal motor weakness and developmental delays. Here, we report detailed biochemical characterization of two novel mutations in CHT1, p.I294T and p.D349N, which we identified in an 11-year-old patient with a history of neonatal respiratory distress, and subsequent hypotonia and global developmental delay. Heterologous expression of each CHT1 mutant in human embryonic kidney cells showed two different mechanisms of reduced protein function. The p.I294T CHT1 mutant transporter function was detectable, but its abundance and half-life were significantly reduced. In contrast, the p.D349N CHT1 mutant was abundantly expressed at the cell membrane, but transporter function was absent. The residual function of the p.I294T CHT1 mutant may explain the non-lethal form of CMS in this patient, and the divergent mechanisms of reduced CHT1 function that we identified may guide future functional studies of the CHT1 myasthenic syndrome. Based on these in vitro studies that provided a diagnosis, treatment with cholinesterase inhibitor together with physical and occupational therapy significantly improved the patient's strength and quality of life., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The identification of amino acid substitutions that both enhance the stability and function of a protein is a key challenge in protein engineering. Technological advances have enabled assaying thousands of protein variants in a single high-throughput experiment, and more recent studies use such data in protein engineering. We present a Global Multi-Mutant Analysis (GMMA) that exploits the presence of multiply-substituted variants to identify individual amino acid substitutions that are beneficial for the stability and function across a large library of protein variants. We have applied GMMA to a previously published experiment reporting on >54,000 variants of green fluorescent protein (GFP), each with known fluorescence output, and each carrying 1-15 amino acid substitutions (Sarkisyan et al., 2016). The GMMA method achieves a good fit to this dataset while being analytically transparent. We show experimentally that the six top-ranking substitutions progressively enhance GFP. More broadly, using only a single experiment as input our analysis recovers nearly all the substitutions previously reported to be beneficial for GFP folding and function. In conclusion, we suggest that large libraries of multiply-substituted variants may provide a unique source of information for protein engineering., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Twinkle is the mammalian helicase vital for replication and integrity of mitochondrial DNA. Over 90 Twinkle helicase disease variants have been linked to progressive external ophthalmoplegia and ataxia neuropathies among other mitochondrial diseases. Despite the biological and clinical importance, Twinkle represents the only remaining component of the human minimal mitochondrial replisome that has yet to be structurally characterized. Here, we present 3-dimensional structures of human Twinkle W315L. Employing cryo-electron microscopy (cryo-EM), we characterize the oligomeric assemblies of human full-length Twinkle W315L, define its multimeric interface, and map clinical variants associated with Twinkle in inherited mitochondrial disease. Cryo-EM, crosslinking-mass spectrometry, and molecular dynamics simulations provide insight into the dynamic movement and molecular consequences of the W315L clinical variant. Collectively, this ensemble of structures outlines a framework for studying Twinkle function in mitochondrial DNA replication and associated disease states.

Approximately 20% of patients with myeloproliferative neoplasms (MPN) harbor mutations in the gene calreticulin (CALR), with 80% of those mutations classified as either type I or type II. While type II CALR-mutant proteins retain many of the Ca2+ binding sites present in the wild-type protein, type I CALR-mutant proteins lose these residues. The functional consequences of this differential loss of Ca2+ binding sites remain unexplored. Here, we show that the loss of Ca2+ binding residues in the type I mutant CALR protein directly impairs its Ca2+ binding ability, which in turn leads to depleted endoplasmic reticulum (ER) Ca2+ and subsequent activation of the IRE1α/XBP1 pathway of the unfolded protein response. Genetic or pharmacologic inhibition of IRE1α/XBP1 signaling induces cell death in type I mutant but not type II mutant or wild-type CALR-expressing cells, and abrogates type I mutant CALR-driven MPN disease progression in vivo., Significance: Current targeted therapies for CALR-mutated MPNs are not curative and fail to differentiate between type I- versus type II-driven disease. To improve treatment strategies, it is critical to identify CALR mutation type-specific vulnerabilities. Here we show that IRE1α/XBP1 represents a unique, targetable dependency specific to type I CALR-mutated MPNs. This article is highlighted in the In This Issue feature, p. 265., (©2022 American Association for Cancer Research.)

Most mutations in the DNA-binding domain (DBD) of p53 inactivate or rescue the protein function interacting with the minor groove of DNA. However, how the conformation changes propagating from the mutation sites result in distinct molecular recognition is still not well understood. As the protein mobility is an intrinsic property encrypted in its primary structure, we examined if different structures of wild-type and mutant p53 core domains display any unique patterns of intrinsic mobility. Normal mode calculation was employed to characterize the collective dynamics of DBD in p53 monomer and tetramer as well as their mutants. Intriguingly, the low-frequency collective motions of DBD show similar patterns between the wild-type protein and the rescued mutants. The analysis on atomic backbone fluctuations and low-frequency vibration mode statistics does further support the correlation between the intrinsic collective motion of DBD and the p53 protein function. The mutations in the DBD influence the low-frequency vibration of the p53 tetramer via the change of the collective motions among its four monomers. These findings thus provide new insights for understanding the physical mechanism of p53 protein structure-function relationship and help find the small molecule drug to modulate protein dynamic for disease therapy., (© 2021 Wiley Periodicals LLC.)

Glycine to serine substitution at position 154 of human αB-crystallin (αB-Cry) is behind the development of cardiomyopathy and late-onset distal myopathy. The current study was conducted with the aim to investigate the structural and functional features of the G154S mutant αB-Cry using various spectroscopic techniques and microscopic analyses. The secondary and tertiary structures of human αB-Cry were preserved mainly in the presence of G154S mutation, but the mutant protein indicated a reduced chaperone-like activity when γ-Cry as its natural partner in eye lenses was the substrate protein. Moreover, a significant reduction in the enzyme refolding ability and in vivo chaperone activity of the mutant protein were observed. Also, the mutant protein displayed reduced conformational stability upon urea-induced denaturation. Both fluorescence and electron microscopic analyses suggested that G154S mutant protein has an increased susceptibility for amyloid fibril formation. Therefore, the pathomechanism of G154S mutation can be explained by its attenuated chaperone function, decreased conformational stability, and increased amyloidogenic propensity. Some of these important changes may also alter the correct interaction of the mutated αB-Cry with its target proteins in myopathy., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Alzheimer's disease displays aggregates of the amyloid-beta (Aβ) peptide in the brain, and there is increasing evidence that cholesterol may contribute to the pathogenesis of the disease. Though many experimental and theoretical studies have focused on the interactions of Aβ oligomers with membrane models containing cholesterol, an understanding of the effect of free cholesterol on small Aβ42 oligomers is not fully established. To address this question, we report on replica exchange with a solute tempering simulation of an Aβ42 trimer with cholesterol and compare it with a previous replica exchange molecular dynamics simulation. We show that the binding hot spots of cholesterol are rather complex, involving hydrophobic residues L17-F20 and L30-M35 with a non-negligible contribution of loop residues D22-K28 and N-terminus residues. We also examine the effects of cholesterol on the trimers of the disease-causing A21G and disease-protective A2T mutations by molecular dynamics simulations. We show that these two mutations moderately impact cholesterol-binding modes. In our REST2 simulations, we find that cholesterol is rarely inserted into aggregates but rather attached as dimers and trimers at the surface of Aβ42 oligomers. We propose that cholesterol acts as a glue to speed up the formation of larger aggregates; this provides a mechanistic link between cholesterol and Alzheimer's disease.

The folding dynamics of proteins is a primary area of interest in protein science. We carried out topological data analysis (TDA) of the folding process of HP35(nle-nle), a double-mutant of the villin headpiece subdomain. Using persistent homology and non-negative matrix factorization, we reduced the dimension of protein structure and investigated the flow in the reduced space. We found this protein has two folding paths, distinguished by the pairings of inter-helix residues. Our analysis showed the excellent performance of TDA in capturing the formation of tertiary structure., (© 2022. The Author(s).)

Ascorbate is an important cellular antioxidant that gets readily oxidized to dehydroascorbate (DHA). Recycling of DHA is therefore paramount in the maintenance of cellular homeostasis and preventing oxidative stress. Dehydroascorbate reductases (DHARs), in conjunction with glutathione (GSH), carry out this vital process in eukaryotes, among which plant DHARs have garnered considerable attention. A detailed kinetic analysis of plant DHARs relative to their human counterparts is, however, lacking. Chloride intracellular channels (HsCLICs) are close homologs of plant DHARs, recently demonstrated to share their enzymatic activity. This study reports the highest turnover rate for a plant DHAR from stress adapted Pennisetum glaucum (PgDHAR). In comparison, HsCLICs 1, 3, and 4 reduced DHA at a significantly lower rate. We further show that the catalytic cysteine from both homologs was susceptible to varying degrees of oxidation, validated by crystal structures and mass-spectrometry. Our findings may have broader implications on crop improvement using pearl millet DHAR vis-à-vis discovery of cancer therapeutics targeting Vitamin-C recycling capability of human CLICs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Mendelian susceptibility to mycobacterial disease (MSMD) is a rare monogenetic disease, which is characterized by susceptibility to some weakly virulent mycobacteria. Here, we explored the pathogenic genes and molecular mechanisms of MSMD patients. We recruited three patients diagnosed with MSMD from two families. Two novel mutations (c.1228A > G, p.K410E and c.2071A > G, p.M691V) in STAT1 gene were identified from two families. The translocation of K410E mutant STAT1 protein into nucleus was not affected. The binding ability between gamma-activating sequence (GAS) and K410E mutant STAT1 protein was significantly reduced, which will reduce the interaction between STAT1 protein with the promoters of target genes. The M691V mutant STAT1 protein cannot translocate into the nucleus after IFN-γ stimulation, which will affect the STAT1 protein form gamma-activating factors (GAF) and bind the GAS in the promoter region of downstream target genes. Taken together, our results showed that the mutation of K410E led to impaired binding of STAT1 to target DNA, and the mutation of M691V prevented the transport of STAT1 into the nucleus, which led to MSMD. Together, we identified two novel mutations (c.1228A > G, p.K410E and c.2071A > G, p.M691V) in STAT1 gene in MSMD patients, and deciphered the molecular mechanism of MSMD caused by STAT1 mutations., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Metabolism has a role in determining the time of pubertal development and fertility. Nonetheless, molecular/cellular pathways linking metabolism/body weight to puberty/reproduction are unknown. The KNDy (Kisspeptin/Neurokinin B/Dynorphin) neurons in the arcuate nucleus of the hypothalamus constitute the GnRH (gonadotropin-releasing hormone) pulse generator. We previously created a mouse model with a whole-body targeted deletion of nescient helix-loop-helix 2 (Nhlh2; N2KO), a class II member of the basic helix-loop-helix family of transcription factors. As this mouse model features pubertal failure and late-onset obesity, we wanted to study whether NHLH2 represents a candidate molecule to link metabolism and puberty in the hypothalamus. Exome sequencing of a large Idiopathic Hypogonadotropic Hypogonadism cohort revealed obese patients with rare sequence variants in NHLH2, which were characterized by in-silico protein analysis, chromatin immunoprecipitation, and luciferase reporter assays. In vitro heterologous expression studies demonstrated that the variant p.R79C impairs Nhlh2 binding to the Mc4r promoter. Furthermore, p.R79C and other variants show impaired transactivation of the human KISS1 promoter. These are the first inactivating human variants that support NHLH2's critical role in human puberty and body weight control. Failure to carry out this function results in the absence of pubertal development and late-onset obesity in humans., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

GRIA3 at Xq25 encodes glutamate ionotropic receptor AMPA type 3 (GluA3), a subunit of postsynaptic glutamate-gated ion channels mediating neurotransmission. Hemizygous loss-of-function (LOF) variants in GRIA3 cause a neurodevelopmental disorder (NDD) in male individuals. Here, we report a gain-of-function (GOF) variant at GRIA3 in a male patient. We identified a hemizygous de novo missense variant in GRIA3 in a boy with an NDD: c.1844C > T (p.Ala615Val) using whole-exome sequencing. His neurological signs, such as hypertonia and hyperreflexia, were opposite to those in previous cases having LOF GRIA3 variants. His seizures and hypertonia were ameliorated by carbamazepine, inhibiting glutamate release from presynapses. Patch-clamp recordings showed that the human GluA3 mutant (p.Ala615Val) had slower desensitization and deactivation kinetics. A fly line expressing a human GluA3 mutant possessing our variant and the Lurcher variant, which makes ion channels leaky, showed developmental defects, while one expressing a mutant possessing either of them did not. Collectively, these results suggest that p.Ala615Val has GOF effects. GRIA3 GOF variants may cause an NDD phenotype distinctive from that of LOF variants, and drugs suppressing glutamatergic neurotransmission may ameliorate this phenotype. This study should help in refining the clinical management of GRIA3-related NDDs., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Induction of bone formation by Wnt ligands is inhibited when sclerostin (Scl), an osteocyte-produced antagonist, binds to its receptors, the low-density lipoprotein receptor-related proteins 5 or 6 (LRP5/6). Recently, it was shown that enhanced inhibition is achieved by Scl binding to the co-receptor LRP4. However, it is not clear if the binding of Scl to LRP4 facilitates Scl binding to LRP5/6 or inhibits the Wnt pathway in an LRP5/6-independent manner. Here, using the yeast display system, we demonstrate that Scl exhibits a stronger binding affinity for LRP4 than for LRP6. Moreover, we found stronger Scl binding to LRP6 in the presence of LRP4. We further show that a Scl mutant (Scl N93A ), which tightly binds LRP4 but not LRP6, does not inhibit the Wnt pathway on its own. We demonstrate that Scl N93A competes with Scl for a common binding site on LRP4 and antagonizes Scl inhibition of the Wnt signaling pathway in osteoblasts in vitro. Finally, we demonstrate that 2 weeks of bi-weekly subcutaneous injections of Scl N93A fused to the fragment crystallizable (Fc) domain of immunoglobulin (Scl N93A Fc), which retains the antagonistic activity of the mutant, significantly increases bone formation rate and enhances trabecular volumetric bone fraction, trabecular number, and bone length in developing mice. Our data show that LRP4 serves as an anchor that facilitates Scl-LRP6 binding and that inhibition of the Wnt pathway by Scl depends on its prior binding to LRP4. We further provide evidence that compounds that inhibit Scl-LRP4 interactions offer a potential strategy to promote anabolic bone functions., (© 2022. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

This work describes the application of a physics-based computational approach to predict the relative thermodynamic stability of protein variants, and evaluates the quantitative accuracy of those predictions compared to experimental data obtained from a diverse set of protein systems assayed at variable pH conditions. Physical stability is a key determinant of the clinical and commercial success of biological therapeutics, vaccines, diagnostics, enzymes and other protein-based products. Although experimental techniques for measuring the impact of amino acid residue mutation on the stability of proteins exist, they tend to be time consuming and costly, hence the need for accurate prediction methods. In contrast to many of the commonly available computational methods for stability prediction, the Free Energy Perturbation approach applied in this paper explicitly accounts for solvent effects and samples conformational dynamics using a rigorous molecular dynamics simulation process. On the entire validation dataset, consisting of 328 single point mutations spread across 14 distinct protein structures, our results show good overall correlation with experiment with an R 2 of 0.65 and a low mean unsigned error of 0.95 kcal/mol. Application of the FEP approach in conjunction with experimental assessment techniques offers opportunities to lower the time and expense of product development and reduce the risk of costly late-stage failures., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

In Brazil, the major vector of arboviruses is Aedes aegypti, which can transmit several alpha and flaviviruses. In this work, a pacifastin protease inhibitor library was constructed and used to select mutants for Ae. aegypti larvae digestive enzymes. The library contained a total of 3.25 × 10 5  cfu with random mutations in the reactive site (P2-P2'). The most successfully selected mutant, TiPI6, a versatile inhibitor, was able to inhibit all three Ae. aegypti larvae proteolytic activities, trypsin-like, chymotrypsin-like and elastase-like activities, with IC 50 values of 0.212 nM, 0.107 nM and 0.109 nM, respectively. In conclusion, the TiPI mutated phage display library was shown to be a useful tool for the selection of an inhibitor of proteolytic activities combined in a mix. TiPI6 is capable of controlling all three digestive enzyme activities present in the larval midgut extract. To our knowledge, this is the first time that one inhibitor containing a Gln at the P1 position showed inhibitory activity against trypsin, chymotrypsin, and elastase-like activities. TiPI6 can be a candidate for further larvicidal studies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The transcription factor c-Myb promotes the proliferation of hematopoietic cells by interacting with the KIX domain of CREB-binding protein; however, its aberrant expression causes leukemia. Therefore, inhibitors of the c-Myb-KIX interaction are potentially useful as antitumor drugs. Since the intrinsically disordered transactivation domain (TAD) of c-Myb binds KIX via a conformational selection mechanism where helix formation precedes binding, stabilizing the helical structure of c-Myb TAD is expected to increase the KIX-binding affinity. Here, to develop an inhibitor of the c-Myb-KIX interaction, we designed mutants of the c-Myb TAD peptide fragment where the helical structure is stabilized, based on theoretical predictions using AGADIR. Three of the four initially designed peptides each had a different Lys-to-Arg substitution on the helix surface opposite the KIX-binding interface. Furthermore, the triple mutant with three Lys-to-Arg substitutions, named RRR, showed a high helical propensity and achieved three-fold higher affinity to KIX than the wild-type TAD with a dissociation constant of 80 nM. Moreover, the RRR inhibitor efficiently competed out the c-Myb-KIX interaction. These results suggest that stabilizing the helical structure based on theoretical predictions, especially by conservative Lys-to-Arg substitutions, is a simple and useful strategy for designing helical peptide inhibitors of protein-protein interactions., (© 2022. The Author(s).)

The influenza virus is an important respiratory pathogen that causes many incidences of diseases and even death each year. One of the primary factors of this virus is the Neuraminidase surface protein, which causes the virus to leave the host cell and spread to new target cells. The main antiviral medication for influenza is designed as a protein inhibitor ligand that prevents further spread of the disease, and eventually relieves the emerged symptoms. The effectiveness of such inhibitory drugs is highly associated with their binding affinity. In this paper, the binding affinity of an herbal ligand of Capsaicin bound to Neuraminidase of the influenza virus is investigated using steered molecular dynamics (SMD) simulation. Since mutations of the virus directly impact the binding affinity of the inhibitory drugs, different mutations were generated by using Mutagenesis module. The rapid spread of infection during the avian influenza A/H5N1 epidemic has raised concerns about far more dangerous consequences if the virus becomes resistant to current drugs. Currently, oseltamivir (Tamiflu), zanamivir (Relenza), pramivir (Rapivab), and laninamivir (Inavir) are increasingly used to treat the flu. However, with the rapid evolution of the virus, some drug-resistant strains are emerging. Therefore, it is very important to seek alternative therapies and identify the roots of drug resistance. Obtained results demonstrated a reduced binding affinity for the applied mutations. This reduction in binding affinity will cause the virus mutation to become resistant to the drug, which will spread the disease and make it more difficult to treat. From a molecular prospect, this decrease in binding affinity is due to the loss of a number of effective bonds between the ligand and the receptor, which occurs with mutations of the wild-type (WT) species. The results of the present study can be used in the rational design of novel drugs that are compatible with specific mutations., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

ATP-sensitive potassium (K-ATP) channels are ubiquitously expressed on the plasma membrane of cells in several organs, including the heart, pancreas, and brain, and they govern a wide range of physiological processes. In pancreatic β-cells, K-ATP channels composed of Kir6.2 and SUR1 play a key role in coupling blood glucose and insulin secretion. A tryptophan residue located at the cytosolic end of the transmembrane helix is highly conserved in eukaryote and prokaryote Kir channels. Any mutation on this amino acid causes a gain of function and neonatal diabetes mellitus. In this study, we have investigated the effect of mutation on this highly conserved residue on a KirBac channel (prokaryotic homolog of mammalian Kir6.2). We provide the crystal structure of the mutant KirBac3.1 W46R (equivalent to W68R in Kir6.2) and its conformational flexibility properties using HDX-MS. In addition, the detailed dynamical view of the mutant during the gating was investigated using the in silico method. Finally, functional assays have been performed. A comparison of important structural determinants for the gating mechanism between the wild type KirBac and the mutant W46R suggests interesting structural and dynamical clues and a mechanism of action of the mutation that leads to the gain of function.

Mutations in the apically located kidney Na-K-2Cl cotransporter NKCC2 cause type I Bartter syndrome, a life-threatening kidney disorder. We previously showed that transport from the ER represents the limiting phase in NKCC2 journey to the cell surface. Yet very little is known about the ER quality control components specific to NKCC2 and its disease-causing mutants. Here, we report the identification of Golgi alpha1, 2-mannosidase IA (ManIA) as a novel binding partner of the immature form of NKCC2. ManIA interaction with NKCC2 takes place mainly at the cis-Golgi network. ManIA coexpression decreased total NKCC2 protein abundance whereas ManIA knock-down produced the opposite effect. Importantly, ManIA coexpression had a more profound effect on NKCC2 folding mutants. Cycloheximide chase assay showed that in cells overexpressing ManIA, NKCC2 stability and maturation are heavily hampered. Deleting the cytoplasmic region of ManIA attenuated its interaction with NKCC2 and inhibited its effect on the maturation of the cotransporter. ManIA-induced reductions in NKCC2 expression were offset by the proteasome inhibitor MG132. Likewise, kifunensine treatment greatly reduced ManIA effect, strongly suggesting that mannose trimming is involved in the enhanced ERAD of the cotransporter. Moreover, depriving ManIA of its catalytic domain fully abolished its effect on NKCC2. In summary, our data demonstrate the presence of a ManIA-mediated ERAD pathway in renal cells promoting retention and degradation of misfolded NKCC2 proteins. They suggest a model whereby Golgi ManIA contributes to ERAD of NKCC2, by promoting the retention, recycling, and ERAD of misfolded proteins that initially escape protein quality control surveillance within the ER.

Deficiency of 21-hydroxylase enzyme (CYP21A2) represents 90% of cases in congenital adrenal hyperplasia (CAH), an autosomal recessive disease caused by defects in cortisol biosynthesis. Computational prediction and functional studies are often the only way to classify variants to understand the links to disease-causing effects. Here we investigated the pathogenicity of uncharacterized variants in the CYP21A2 gene reported in Brazilian and Portuguese populations. Physicochemical alterations, residue conservation, and effect on protein structure were accessed by computational analysis. The enzymatic performance was obtained by functional assay with the wild-type and mutant CYP21A2 proteins expressed in HEK293 cells. Computational analysis showed that p.W202R, p.E352V, and p.R484L have severely impaired the protein structure, while p.P35L, p.L199P, and p.P433L have moderate effects. The p.W202R, p.E352V, p.P433L, and p.R484L variants showed residual 21OH activity consistent with the simple virilizing phenotype. The p.P35L and p.L199P variants showed partial 21OH efficiency associated with the non-classical phenotype. Additionally, p.W202R, p.E352V, and p.R484L also modified the protein expression level. We have determined how the selected CYP21A2 gene mutations affect the 21OH activity through structural and activity alteration contributing to the future diagnosis and management of CYP21A2 deficiency.

We examined keratin aggregate formation and the possible mechanisms involved. With this aim, we observed the effect that different ratios between mutant and wild-type keratins expressed in cultured keratinocytes may have on aggregate formation in vitro, as well as how keratin aggregate formation affects the mechanical properties of cells at the cell cortex. To this end we prepared clones with expression rates as close as possible to 25%, 50% and 100% of the EGFP-K14 proteins (either WT or R125P and V270M mutants). Our results showed that only in the case of the 25% EGFP-K14 R125P mutant significant differences could be seen. Namely, we observed in this case the largest accumulation of keratin aggregates and a significant reduction in cell stiffness. To gain insight into the possible mechanisms behind this observation, we extended our previous mathematical model of keratin dynamics by implementing a more complex reaction network that considers the coexistence of wild-type and mutant keratins in the cell. The new model, consisting of a set of coupled, non-linear, ordinary differential equations, allowed us to draw conclusions regarding the relative amounts of intermediate filaments and aggregates in cells, and suggested that aggregate formation by asymmetric binding between wild-type and mutant keratins could explain the data obtained on cells grown in culture., Competing Interests: The authors have declared that no competing interests exist.

Accidents involving Brown spiders are reported throughout the world. In the venom, the major toxins involved in the deleterious effects are phospholipases D (PLDs). In this work, recombinant mutated phospholipases D from three endemic species medically relevant in South America (Loxosceles intermedia, L. laeta and L. gaucho) were tested as antigens in a vaccination protocol. In such isoforms, key amino acid residues involved in catalysis, magnesium-ion coordination, and binding to substrates were replaced by Alanine (H12A-H47A, E32A-D34A and W230A). These mutations eliminated the phospholipase activity and reduced the generation of skin necrosis and edema to residual levels. Molecular modeling of mutated isoforms indicated that the three-dimensional structures, topologies, and surface charges did not undergo significant changes. Mutated isoforms were recognized by sera against the crude venoms. Vaccination protocols in rabbits using mutated isoforms generated a serum that recognized the native PLDs of crude venoms and neutralized dermonecrosis and edema induced by L. intermedia venom. Vaccination of mice prevented the lethal effects of L. intermedia crude venom. Furthermore, vaccination of rabbits prevented the cutaneous lesion triggered by the three venoms. These results indicate a great potential for mutated recombinant PLDs to be employed as antigens in developing protective vaccines for Loxoscelism., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The aberrant kinase activity of RET (rearranged during transfection), a transmembrane tyrosine kinase, is associated with human cancer. A point mutation caused by the replacement of solvent-front hydrophilic S904, located on the activation loop (A-loop), with a bulky hydrophobic phenylalanine residue can induce resistance to the type I kinase inhibitor vandetanib. A possible mechanism of this drug resistance is the release of a cis-autoinhibited conformation of RET for autophosphorylation, which activates RET kinase. Because the association between S904F mutation and enhanced autophosphorylation is unclear, we conducted molecular modeling analysis to compare unphosphorylated apo wild-type and S904F mutant structures. The structural compactness of the A-loop promoted ATP binding. When the A-loop is extended, the αC helix moves toward the glycine-rich loop, resulting in the protrusion of F735. The extruded F735 connects with E734 and R912 and constrains the ATP pocket entrance. Contrarily, a contracted A-loop pulls the αC helix away from the glycine-rich loop, burying F734 and making the ATP pocket accessible. The mutated F904 stabilizes the contracted A-loop and releases the autoinhibited conformation of RET, thereby facilitating autophosphorylation. We also simulated two ATP-bound systems. The binding free energies of ATP, estimated through the molecular mechanics with a generalized Born and surface area solvation approach, revealed that the S904F mutant was bound more tightly than was the wild type with the ATP. The findings support the premise of autophosphorylation promotion in the S904F mutant., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The Asp233-Asp246 pair is highly conserved in Class A β-lactamases, which hydrolyze β-lactam antibiotics. Here, we characterize its function using CTX-M-14 β-lactamase. The D233N mutant displayed decreased activity that is substrate-dependent, with reductions in k cat /K m ranging from 20% for nitrocefin to 6-fold for cefotaxime. In comparison, the mutation reduced the binding of a known reversible inhibitor by 10-fold. The mutant structures showed movement of the 213-219 loop and the loss of the Thr216-Thr235 hydrogen bond, which was restored by inhibitor binding. Mutagenesis of Thr216 further highlighted its contribution to CTX-M activity. These results demonstrate the importance of the aspartate pair to CTX-M hydrolysis of substrates with bulky side chains, while suggesting increased protein flexibility as a means to evolve drug resistance., (© 2021 Federation of European Biochemical Societies.)

The wild-type protein p53 plays a key role in preventing the formation of neoplasms by controlling cell growth. However, in more than a half of all cancers, the TP53 gene has missense mutations that appear during tumorigenesis. In most cases, the mutated gene encodes a full-length protein with the substitution of a single amino acid, resulting in structural and functional changes and acquiring an oncogenic role. This dual role of the wild-type protein and the mutated isoforms is also evident in the regulation of the redox state of the cell, with antioxidant and prooxidant functions, respectively. In this review, we introduce a new concept of the p53 protein by discussing its sensitivity to the cellular redox state. In particular, we focus on the discussion of structural and functional changes following post-translational modifications of redox-sensitive cysteine residues, which are also responsible for interacting with zinc ions for proper structural folding. We will also discuss therapeutic opportunities using small molecules targeting cysteines capable of modifying the structure and function of the p53 mutant isoforms in view of possible anticancer therapies for patients possessing the mutation in the TP53 gene.

Developmental and epileptic encephalopathies (DEE) are rare and serious neurological disorders characterized by severe epilepsy with refractory seizures and a significant developmental delay. Recently, DEE73 was linked to genetic alterations of the RNF13 gene, which convert positions 311 or 312 in the RNF13 protein from leucine to serine or proline, respectively (L311S and L312P). Using a fluorescence microscopy approach to investigate the molecular and cellular mechanisms affected by RNF13 protein variants, the current study shows that wild-type RNF13 localizes extensively with endosomes and lysosomes, while L311S and L312P do not extensively colocalize with the lysosomal marker Lamp1. Our results show that RNF13 L311S and L312P proteins affect the size of endosomal vesicles along with the temporal and spatial progression of fluorescently labeled epidermal growth factor, but not transferrin, in the endolysosomal system. Furthermore, GST-pulldown and co-immunoprecipitation show that RNF13 variants disrupt association with AP-3 complex. Knockdown of AP-3 complex subunit AP3D1 alters the lysosomal localization of wild-type RNF13 and similarly affects the size of endosomal vesicles. Importantly, our study provides a first step toward understanding the cellular and molecular mechanism altered by DEE73-associated genetic variations of RNF13.

In human αB-crystallin or HspB5, the substitution of arginine residue at position 157 with histidine has been reported to cause cardiomyopathy. In this study, the impact of R157H mutation on the structure, stability and functional properties of human αB-crystallin was investigated using a variety of spectroscopic techniques and microscopic analyses. Our spectroscopic analyses revealed that this mutation has a negligible impact on the secondary and tertiary structures of HspB5 but its quaternary structure underwent fundamental changes. Although the chemical stability of the mutant protein remained largely unchanged, the differential scanning calorimetry (DSC) measurement suggested that its thermal stability was reduced. As examined with transmission electron microscopy, αB-crystallin and its mutant indicated a similar tendency for the amyloid fibril formation under thermochemical stress. Dynamic light scattering (DLS) analysis suggested important changes in the quaternary (oligomeric) structures of the mutant protein as compared with the native protein counterpart. Also, the mutant protein indicated an improved chaperone-like activity under in vitro assessment. In a pH-dependent manner, the side chains of arginine and histidine have different capabilities for establishing hydrogen bonds and electrostatic interaction (salt bridge) and this variation may be sufficient to produce the larger changes that ultimately alter the interaction of this protein with other target proteins. Overall, the pathogenic contribution of this mutation in cardiomyopathy can be explained by its role in quaternary structure/stability alteration of the mutated protein., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

The EccC enzyme of ESX-1 system contains (i) a membrane bound Rv3870 with single ATPase domain and (ii) a cytoplasmic Rv3871 with two ATPase domains and involved in secretion of ESAT6/CFP10 factor out of the cell. In current study, we have structurally and biochemically characterized the ATPase domain (442-747 residues) of Rv3870 enzyme. The ΔRv3870 eluted as oligomer (~813 kDa) from Superdex 200 (16/60) column, as identified based on molecular mass standard and dynamics light scattering. The SAXS analysis yielded a tetrameric ring envelope of ΔRv3870, quite consistent to dynamic light scattering data. The ΔRv3870 exhibited ATPase activity having kinetic parameters, K m  ~ 100 ± 40 μM, k cat  ~ 1.81 ± 0.27 min -1 and V max  ~ 54.41 μM/min/mg. ATPase activity using nine ΔRv3870 mutants showed 70-91% decrease in catalytic efficiency of the enzyme. ΔRv3870 binds Rv3871 with K D  ~ 484.0 ± 10.3 nM and its catalytic efficiency is enhanced ~6.7-fold in presence of Rv3871. CD data revealed the high T M  ~ 82.2 ± 0.5 °C for ΔRv3870 and enhanced in presence of ATP + Mg 2+ , as observed in dynamics simulation on ΔRv3870 hexameric models. Overall, our structural and biochemical studies on ΔRv3870 have explained the mechanism, which will contribute in development of antivirulence inhibitors against M. tuberculosis., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Congenital cataracts, which are genetically heterogeneous eye disorders, lead to visual impairment in childhood. In our previous study, we identified a novel mutation in exon 4 of the CRYBA1/BA3 gene, which resulted in the deletion of a highly conserved glycine at codon 91 (G91del) and perinuclear zonular cataract. The G91del variant is one of the most frequent pathogenic mutations in CRYBA1/BA3; however, its pathogenic mechanism remains unclear. In this study, we purified βA3-crystallin and the βA3-G91del variant. βA3-G91del was prone to proteolysis and exhibited very low solubility and low structural stability. Next, we constructed a CRYBA1/BA3 mutant cell model and observed that G91del mutant proteins were more sensitive to environmental stress and prone to form aggregates. Size-exclusion chromatography and molecular dynamics simulation showed that the G91del mutation impaired the ability of βA3 to form homo-oligomers. In addition, the protein folding process of βA3-G91del was complicated and showed more intermediate states, resulting in amyloid fiber aggregation and induction of cellular apoptosis. Finally, we investigated intervention strategies for congenital cataract caused by the CRYBA1/A3-G91del variant. The addition of lanosterol reversed the negative effects of the G91del mutation under external stress. This study may help explore potential treatment strategies for related cataracts., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Protein misfolding and aggregation are implicated in many neurodegenerative diseases. One of these diseases is Huntington's, which is caused by increased glutamine-encoding trinucleotide repeats within the Huntingtin gene. Like other misfolded proteins, mutated Huntingtin proteins with polyglutamine expansions are prone to aggregation. Misfolded proteins exist as soluble monomers, small aggregates, or as large insoluble inclusion bodies. Misfolded protein aggregates are believed to be cytotoxic by stressing the protein degradation machinery, disrupting membrane structure, or sequestering other proteins. We recently showed that expression of misfolded proteins lowers cellular free ubiquitin levels, which compromises the protein degradation machinery. Therefore, the efficient degradation of misfolded proteins is critical to preserve cell health. Cells employ two major mechanisms to degrade misfolded proteins. The first is the ubiquitin-proteasome system (UPS), which ubiquitinates and degrades misfolded proteins with the assistance of segregase Cdc48/p97. The UPS pathway is mainly responsible for the clearance of misfolded proteins present as monomers or smaller aggregates. The second pathway is macroautophagy/autophagy, in which protein aggregates or inclusion bodies are recruited into an autophagosome before transport to the vacuole/lysosome for degradation. This review is focused on the current understanding of the cytotoxicity of misfolded proteins as well as their clearance pathways, with a particular emphasis on mutant Huntingtin.

Photosynthetic pigment-protein complexes control local chlorophyll (Chl) transition frequencies through a variety of electrostatic and steric forces. Site-directed mutations can modify this local spectroscopic tuning, providing critical insight into native photosynthetic functions and offering the tantalizing prospect of creating rationally designed Chl proteins with customized optical properties. Unfortunately, at present, no proven methods exist for reliably predicting mutation-induced frequency shifts in advance, limiting the method's utility for quantitative applications. Here, we address this challenge by constructing a series of point mutants in the water-soluble chlorophyll protein of Lepidium virginicum and using them to test the reliability of a simple computational protocol for mutation-induced site energy shifts. The protocol uses molecular dynamics to prepare mutant protein structures and the charge density coupling model of Adolphs et al. [Photosynth. Res. 95, 197-209 (2008)] for site energy prediction; a graphical interface that implements the protocol automatically is published online at http://nanohub.org/tools/pigmenthunter. With the exception of a single outlier (presumably due to unexpected structural changes), we find that the calculated frequency shifts match the experiment remarkably well, with an average error of 1.6 nm over a 9 nm spread in wavelengths. We anticipate that the accuracy of the method can be improved in the future with more advanced sampling of mutant protein structures.

TP53 plays critical roles in maintaining genome stability. Deleterious genetic variants damage the function of TP53, causing genome instability and increased cancer risk. Of the large quantity of genetic variants identified in TP53, however, many remain functionally unclassified as variants of unknown significance (VUS) due to the lack of evidence. This is reflected by the presence of 749 (42%) VUS of the 1785 germline variants collected in the ClinVar database. In this study, we addressed the deleteriousness of TP53 missense VUS. Utilizing the protein structure-based Ramachandran Plot-Molecular Dynamics Simulation (RPMDS) method that we developed, we measured the effects of missense VUS on TP53 structural stability. Of the 340 missense VUS tested, we observed deleterious evidence for 193 VUS, as reflected by the TP53 structural changes caused by the VUS-substituted residues. We compared the results from RPMDS with those from other in silico methods and observed higher specificity of RPMDS in classification of TP53 missense VUS than these methods. Data from our current study address a long-standing challenge in classifying the missense VUS in TP53, one of the most important tumor suppressor genes.

Lon is an ATP-dependent protease belonging to the "ATPase associated with diverse cellular activities" (AAA+) protein family. In humans, Lon is translated as a precursor and imported into the mitochondria matrix through deletion of the first 114 amino acid residues. In mice, embryonic knockout of lon is lethal. In humans, some dysfunctional lon mutations are tolerated but they cause a developmental disorder known as the CODAS syndrome. To gain a better understanding on the enzymology of human mitochondrial Lon, this study compares the structure-function relationship of the WT versus one of the CODAS mutants R721G to identify the mechanistic features in Lon catalysis that are affected. To this end, steady-state kinetics were used to quantify the difference in ATPase and ATP-dependent peptidase activities between WT and R721G. The K m values for the intrinsic as well as protein-stimulated ATPase were increased whereas the k cat value for ATP-dependent peptidase activity was decreased in the R721G mutant. The mutant protease also displayed substrate inhibition kinetics. In vitro studies revealed that R721G did not degrade the endogenous mitochondrial Lon substrate pyruvate dehydrogenase kinase isoform 4 (PDK4) effectively like WT hLon. Furthermore, the pyruvate dehydrogenase complex (PDH) protected PDK4 from hLon degradation. Using hydrogen deuterium exchange/mass spectrometry and negative stain electron microscopy, structural perturbations associated with the R721G mutation were identified. To validate the in vitro findings under a physiologically relevant condition, the intrinsic stability as well as proteolytic activity of WT versus R721G mutant towards PDK 4 were compared in cell lysates prepared from immortalized B lymphocytes expressing the respective protease. The lifetime of PDK4 is longer in the mutant cells, but the lifetime of Lon protein is longer in the WT cells, which corroborate the in vitro structure-functional relationship findings., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Asthenozoospermia is the most common cause of male infertility. Dynein protein arms play a crucial role in the motility of both the cilia and flagella, and defects in these proteins generally impair the axoneme structure and cause primary ciliary dyskinesia. But relatively little is known about the influence of dynein protein arm defects on sperm flagella function. Here, we recruited 85 infertile patients with idiopathic asthenozoospermia and identified bi-allelic mutations in DNAH7 (NM_018897.3) from three patients using whole-exome sequencing. These variants are rare, highly pathogenic, and very conserved. The spermatozoa from the patients with DNAH7 bi-allelic mutations showed specific losses in the inner dynein arms. The expression of DNAH7 in the spermatozoa from the DNAH7-defective patients was significantly decreased, but these patients were able to have their children via intra-cytoplasmic sperm injection treatment. Our study is the first to demonstrate that bi-allelic mutations in DNAH7 may impair the integrality of axoneme structure, affect sperm motility, and cause asthenozoospermia in humans. These findings may extend the spectrum of etiological genes and provide new clues for the diagnosis and treatment of patients with asthenozoospermia., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)