Deep computational phenotyping of genomic variants impacting the SET domain of KMT2C reveal molecular mechanisms for their dysfunction.

Introduction: Kleefstra Syndrome type 2 (KLEFS-2) is a genetic, neurodevelopmental disorder characterized by intellectual disability, infantile hypotonia, severe expressive language delay, and characteristic facial appearance, with a spectrum of other distinct clinical manifestations. Pathogenic mutations in the epigenetic modifier type 2 lysine methyltransferase KMT2C have been identified to be causative in KLEFS-2 individuals. Methods: This work reports a translational genomic study that applies a multidimensional computational approach for deep variant phenotyping, combining conventional genomic analyses, advanced protein bioinformatics, computational biophysics, biochemistry, and biostatistics-based modeling. We use standard variant annotation, paralog annotation analyses, molecular mechanics, and molecular dynamics simulations to evaluate damaging scores and provide potential mechanisms underlying KMT2C variant dysfunction. Results: We integrated data derived from the structure and dynamics of KMT2C to classify variants into SV (Structural Variant), DV (Dynamic Variant), SDV (Structural and Dynamic Variant), and VUS (Variant of Uncertain Significance). When compared with controls, these variants show values reflecting alterations in molecular fitness in both structure and dynamics. Discussion: We demonstrate that our 3D models for KMT2C variants suggest distinct mechanisms that lead to their imbalance and are not predictable from sequence alone. Thus, the missense variants studied here cause destabilizing effects on KMT2C function by different biophysical and biochemical mechanisms which we adeptly describe. This new knowledge extends our understanding of how variations in the KMT2C gene cause the dysfunction of its methyltransferase enzyme product, thereby bearing significant biomedical relevance for carriers of KLEFS2-associated genomic mutations.

Beyond structural bioinformatics for genomics with dynamics characterization of an expanded KRAS mutational landscape.

Current capabilities in genomic sequencing outpace functional interpretations. Our previous work showed that 3D protein structure calculations enhance mechanistic understanding of genetic variation in sequenced tumors and patients with rare diseases. The KRAS GTPase is among the critical genetic factors driving cancer and germline conditions. Because KRAS-altered tumors frequently harbor one of three classic hotspot mutations, nearly all studies have focused on these mutations, leaving significant functional ambiguity across the broader KRAS genomic landscape observed in cancer and non-cancer diseases. Herein, we extend structural bioinformatics with molecular simulations to study an expanded landscape of 86 KRAS mutations. We identify multiple coordinated changes strongly associated with experimentally established KRAS biophysical and biochemical properties. The patterns we observe span hotspot and non-hotspot alterations, which can all dysregulate Switch regions, producing mutation-restricted conformations with different effector binding propensities. We experimentally measured mutation thermostability and identified shared and distinct patterns with simulations. Our results indicate mutation-specific conformations, which show potential for future research into how these alterations reverberate into different molecular and cellular functions. The data we present is not predictable using current genomic tools, demonstrating the added functional information derived from molecular simulations for interpreting human genetic variation.

RAG genomic variation causes autoimmune diseases through specific structure-based mechanisms of enzyme dysregulation.

Interpreting genetic changes observed in individual patients is a critical challenge. The array of immune deficiency syndromes is typically caused by genetic variation unique to individuals. Therefore, new approaches are needed to interpret functional variation and accelerate genomics interpretation. We constructed the first full-length structural model of human RAG recombinase across four functional states of the recombination process. We functionally tested 182 clinically observed RAG missense mutations. These experiments revealed dysfunction due to recombinase dysfunction and altered chromatin interactions. Structural modeling identified mechanical and energetic roles for each mutation. We built regression models for RAG1 (R2 = 0.91) and RAG2 (R2 = 0.97) to predict RAG activity changes. We applied our model to 711 additional RAG variants observed in population studies and identified a subset that may impair RAG function. Thus, we demonstrated a fundamental advance in the mechanistic interpretation of human genetic variations spanning from rare and undiagnosed diseases to population health.

Beyond Structural Bioinformatics for Genomics with Dynamics Characterization of an Expanded KRAS Mutational Landscape.

Current capabilities in genomic sequencing outpace functional interpretations. Our previous work showed that 3D protein structure calculations enhance mechanistic understanding of genetic variation in sequenced tumors and patients with rare diseases. The KRAS GTPase is among the critical genetic factors driving cancer and germline conditions. Because KRAS-altered tumors frequently harbor one of three classic hotspot mutations, nearly all studies have focused on these mutations, leaving significant functional ambiguity across the broader KRAS genomic landscape observed in cancer and non-cancer diseases. Herein, we extend structural bioinformatics with molecular simulations to study an expanded landscape of 86 KRAS mutations. We identify multiple coordinated changes strongly associated with experimentally established KRAS biophysical and biochemical properties. The patterns we observe span hotspot and non-hotspot alterations, which can all dysregulate Switch regions, producing mutation-restricted conformations with different effector binding propensities. We experimentally measured mutation thermostability and identified shared and distinct patterns with simulations. Our results indicate mutation-specific conformations which show potential for future research into how these alterations reverberate into different molecular and cellular functions. The data we present is not predictable using current genomic tools, demonstrating the added functional information derived from molecular simulations for interpreting human genetic variation.

Correction: Haque et al. Marine Natural Products in Clinical Use. Mar. Drugs 2022, 20, 528.

The authors would like to make corrections to a recently published paper [...].

Marine Natural Products in Clinical Use.

Marine natural products are potent and promising sources of drugs among other natural products of plant, animal, and microbial origin. To date, 20 drugs from marine sources are in clinical use. Most approved marine compounds are antineoplastic, but some are also used for chronic neuropathic pain, for heparin overdosage, as haptens and vaccine carriers, and for omega-3 fatty-acid supplementation in the diet. Marine drugs have diverse structural characteristics and mechanisms of action. A considerable increase in the number of marine drugs approved for clinical use has occurred in the past few decades, which may be attributed to increasing research on marine compounds in laboratories across the world. In the present manuscript, we comprehensively studied all marine drugs that have been successfully used in the clinic. Researchers and clinicians are hopeful to discover many more drugs, as a large number of marine natural compounds are being investigated in preclinical and clinical studies.

Identification of a new TRAF6 inhibitor for the treatment of hepatocellular carcinoma.

Tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) is an E3 ubiquitin ligase that plays a crucial role in signal transduction. Previous studies have demonstrated that TRAF6 is overexpressed in hepatocellular carcinoma (HCC) and that TRAF6 knockdown dramatically attenuates tumor cell growth. Thus, TRAF6 may represent a potential therapeutic target for the treatment of HCC. Herein, we identified bis (4-hydroxy-3,5-dimethylphenyl) sulfone (TMBPS) as a novel inhibitor that can directly bind to and downregulate the level of TRAF6. In vitro experimental results showed that TMBPS arrests the cell cycle in the G2/M phase by inactivating the protein kinase B (AKT) and extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathways and induces apoptosis by activating the p38/mitogen-activated protein kinase (MAPK) signaling pathway. In addition, TMBPS exhibited significant tumor growth inhibition in mouse xenograft models. In summary, our findings offer a proof-of-concept for the use of TMBPS as a novel chemotherapy drug for the prevention or treatment of HCC.

Puromycin, a selective inhibitor of PSA acts as a substrate for other M1 family aminopeptidases: Biochemical and structural basis.

Puromycin sensitive aminopeptidase (PSA or NPEPPS) is a M1 class aminopeptidase is selectively inhibited by the natural product puromycin, an aminonucleoside antibiotic produced by the bacterium Streptomyces alboniger. The molecular basis for this selective inhibition has not been understood well. Here, we report the basis for selectivity of puromycin using biochemical, structural and molecular modeling tools on four different M1 family enzymes including human PSA. Except for PSA, the other three enzymes were not inhibited. Instead, the peptide bond in the puromycin is hydrolyzed to O-methyl-L-tyrosine (OMT) and puromycin aminonucleoside (PAN). Neither of the hydrolyzed products, individually or together inhibit any of the four enzymes. Crystal structure of ePepN using crystals that are incubated with puromycin contained the hydrolyzed products instead of intact puromycin. On the other hand, intact puromycin molecule was observed in the crystal structure of the inactive mutant ePepN (E298A)-puromycin complex. Surprisingly, puromycin does not enter the active site of the mutant enzyme but binds near the entrance. Comparison of puromycin binding region in ePepN mutant enzyme and molecular modeling studies suggest that PSA might be inhibited by similar mode of binding there by blocking the entrance of the active site.

Popular Computational Tools Used for miRNA Prediction and Their Future Development Prospects.

MicroRNAs (miRNAs) are 19-24 nucleotide (nt)-long noncoding, single-stranded RNA molecules that play significant roles in regulating the gene expression, growth, and development of plants and animals. From the year that miRNAs were first discovered until the beginning of the twenty-first century, researchers used experimental methods such as cloning and sequencing to identify new miRNAs and their roles in the posttranscriptional regulation of protein synthesis. Later, in the early 2000s, informatics approaches to the discovery of new miRNAs began to be implemented. With increasing knowledge about miRNA, more efficient algorithms have been developed for computational miRNA prediction. The miRNA research community, hoping for greater coverage and faster results, has shifted from cumbersome and expensive traditional experimental approaches to computational approaches. These computational methods started with homology-based comparisons of known miRNAs with orthologs in the genomes of other species; this method could identify a known miRNA in new species. Second-generation sequencing and next-generation sequencing of mRNA at different developmental stages and in specific tissues, in combination with a better search and alignment algorithm, have accelerated the process of predicting novel miRNAs in a particular species. Using the accumulated annotated miRNA sequence information, researchers have been able to design ab initio algorithms for miRNA prediction independent of genome sequence knowledge. Here, the methods recently used for miRNA computational prediction are summarized and classified into the following four categories: homology-based, target-based, scoring-based, and machine-learning-based approaches. Finally, the future developmental directions of miRNA prediction methods are discussed.

Discovery of natural product ovalicin sensitive type 1 methionine aminopeptidases: molecular and structural basis.

Natural product ovalicin and its synthetic derivative TNP-470 have been extensively studied for their antiangiogenic property, and the later reached phase 3 clinical trials. They covalently modify the conserved histidine in Type 2 methionine aminopeptidases (MetAPs) at nanomolar concentrations. Even though a similar mechanism is possible in Type 1 human MetAP, it is inhibited only at millimolar concentration. In this study, we have discovered two Type 1 wild-type MetAPs (Streptococcus pneumoniae and Enterococcus faecalis) that are inhibited at low micromolar to nanomolar concentrations and established the molecular mechanism. F309 in the active site of Type 1 human MetAP (HsMetAP1b) seems to be the key to the resistance, while newly identified ovalicin sensitive Type 1 MetAPs have a methionine or isoleucine at this position. Type 2 human MetAP (HsMetAP2) also has isoleucine (I338) in the analogous position. Ovalicin inhibited F309M and F309I mutants of human MetAP1b at low micromolar concentration. Molecular dynamics simulations suggest that ovalicin is not stably placed in the active site of wild-type MetAP1b before the covalent modification. In the case of F309M mutant and human Type 2 MetAP, molecule spends more time in the active site providing time for covalent modification.

Discovery of a new class of type 1 methionine aminopeptidases that have relaxed substrate specificity.

Methionine aminopeptidases (MetAPs) are a class of enzymes evolved to cleave initiator methionine in 60-70% of the total cellular proteins in all living cells. Based on their sequence differences, they are classified into Type 1 and Type 2. Type 1 is further divided into Type 1a, 1a', 1b, 1c and 1d. Irrespective of various classifications, all MetAPs reported till date displayed hydrolytic activity against peptides that contain only methionine on the N-terminus. A cysteine at the top of the active site in all the Type 1 structures is reported to be critical for the specificity. Mutation of this cysteine to serine or asparagine leads to loss of specificity. In the present study, we have identified a class of MetAPs in some of the proteobacteria that have an asparagine at this site. Most of the proteobacteria that contain MetAP1n are pathogenic in nature. Biochemical and structural studies on two proteins, one from each of V. coralliilyticus and K. pneumoniae confirm that these enzymes cleave leucine in addition to methionine. Crystallographic and homology modeling studies suggest that relaxed substrate specificity of this new class of enzymes could be due to the increased flexibility in the active site. Since this new class has an asparagine at the critical position that probably contributes for the relaxed substrate specificity and also differentiates them from other Type 1 MetAPs, we classified them as Type 1n.

Discovery, Structural and Biochemical Studies of a rare Glu/Asp Specific M1 Class Aminopeptidase from Legionella pneumophila.

Aminopeptidases catalyze the hydrolysis of amino acids from the N-terminus of protein or peptide substrates. M1 family aminopeptidases are important for the pathogenicity of bacteria and play critical role in many physiological processes such as protein maturation, regulation of peptide hormone levels in humans. Most of the M1 family aminopeptidases reported till date display broad substrates specificity, mostly specific to basic and hydrophobic residues. In the current study we report the discovery of a novel M1 class aminopeptidase from Legionella pneumophila (LePepA), which cleaves only acidic residues. Biochemical and structural studies reveal that the S1 pocket is polar and positively charged. Bioinformatic analysis suggests that such active site is unique to only Legionella species and probably evolved for special needs of the microbe. Given its specific activity, LePepA could be useful in specific biotechnological applications.

Binding orientation and interaction of bile salt in its ternary complex with pancreatic lipase-colipase system.

The interfacial activity of pancreatic lipases (PL) depends on the presence of colipase and bile salt. The activity of PL is inhibited by micellar concentrations of bile salt which can be restored by the addition of colipase. Though the formation of 1:1:1 tertiary complex by lipase-colipase-bile salt micelle is well accepted, the residue-level interactions between lipase-colipase and bile salt are yet to be clearly understood. Molecular dynamic simulations of lipase-colipase complex, lipase and colipase were performed in the presence of a model bile salt, sodium taurocholate (NaTC), at its near-CMC and supra-micellar concentrations. From the interactions obtained from the molecular dynamic simulations, the ternary complex was modelled and compared with earlier reports. The analysis suggested that a micelle of NaTC consisting of nine monomers was formed at the concave groove between lipase and colipase chain and it mainly interacted with the fourth finger of colipase. This complex was mainly stabilized by van der Waals interactions. Interestingly, the C-terminal domain of lipase which holds the colipase did not show any significant role in formation or stabilization of NaTC micelle.

Analysing the microenvironment of 2-p-toluidinylnaphthalene-6-sulfonate (TNS) in solvents and in different conformational states of proteins in relation to its fluorescence properties: a computational study.

Characterization of different conformational states of proteins is essential to understand their stability and activity. Biophysical techniques aid in analysing these conformational states and molecular fluorescence is one of the most reliable and quickly accessible methods. Apart from the intrinsic fluorescence of proteins, external fluorescence dyes such as TNS, ANS, nile red and thioflavin are also used to characterize partially unfolded, aggregated and fibrillar states of proteins, though their exact molecular-level interactions with proteins are yet to be completely unravelled. The present study attempts to investigate the binding of TNS molecules on different conformational states of proteins. Unconstrained molecular dynamics simulation of 50 molecules of TNS with the native state of BSA, native and two partially unfolded states of RNase A and α-lactalbumin in water was carried out. Dynamics simulation of TNS alone in different solvents such as water, ethanol, DMF and DMSO was also performed. Binding environments in all the proteins and the solvents were analysed in terms of H-bonding interactions, order of contacts, amino acid specificity and conformational changes of TNS, and correlated with experimentally observed fluorescence changes of the dye. The results suggest that TNS forms aggregates in water whereas in non-aqueous solvents the order of aggregates is lower which might result in an enhancement of its fluorescence intensity. Further, TNS preferably interacts with basic and aromatic amino acid residues of the proteins. In RNase A and α-lactalbumin, most of the TNS molecules tend to form aggregates even with the unfolded conformations of the proteins. However in BSA, the number of aggregated TNS molecules is less and TNS molecules in monomeric form are found in the hydrophobic crevices of the protein. This might result in an enhancement of the fluorescence in BSA compared to the other proteins. The distributions of angles and dihedrals of TNS in different environments suggest that the bending movement between the naphthyl and tolyl rings is constrained whereas significant planar rotations could be observed both in solvents and in protein-bound states.

Kinetics of protein fibril formation: Methods and mechanisms.

Amyloid fibril formation is a self-assembly reaction induced by favourable conformational changes of proteins leading to a stable, structurally organized aggregates. The deposition of stable protein fibrils in organs and tissues results in many diseases which are generally referred as amyloidosis. Though different disease conditions originate from sequentially and structurally different proteins, their fibrillar forms share common structural features. In vitro, fibril structure and kinetic pathway are investigated by using spectroscopic (fluorescence, circular dichroism, crystallography and solid state-NMR) and microscopic techniques. The kinetics of fibril formation is analysed using different mechanisms to understand the microscopic processes involved in the fibrillation reaction. This review discusses the assumptions, mechanisms, and limitations of some of the widely applied kinetic equations. Understanding of these equations would help to quantify the effect of the different microscopic process on the overall fibrillation kinetics which could aid in designing appropriate molecules to intervene in the aggregation process at different stages.

Lid dynamics of porcine pancreatic lipase in non-aqueous solvents.

BACKGROUND: Understanding the dynamics of enzymes in organic solvents has wider implications on their industrial applications. Pancreatic lipases, which show activity in their lid open-state, demonstrate enhanced activity in organic solvents at higher temperatures. However, the lid dynamics of pancreatic lipases in non-aqueous environment is yet to be clearly understood. METHODS: Dynamics of porcine pancreatic lipase (PPL) in open and closed conformations was followed in ethanol, toluene, and octanol using molecular simulation methods. In silico double mutant D250V and E254L of PPL (PPLmut-Cl) was created and its lid opening dynamics in water and in octanol was analyzed. RESULTS: PPL showed increase in solvent accessible surface area and decrease in packing density as the polarity of the surrounded solvent decreased. Breaking the interactions between D250-Y115, and D250-E254 in PPLmut-Cl directed the lid to attain open-state conformation. Major energy barriers during the lid movement in water and in octanol were identified. Also, the trajectories of lid movement were found to be different in these solvents. CONCLUSIONS: Only the double mutant at higher temperature showed lid opening movement suggesting the essential role of the three residues in holding the lid in closed conformation. The lid opening dynamics was faster in octanol than water suggesting that non-polar solvents favor open conformation of the lid. GENERAL SIGNIFICANCE: This study identifies important interactions between the lid and the residues in domain 1 which possibly keeps the lid in closed conformation. Also, it explains the rearrangements of residue-residue interactions during lid opening movement in water and in octanol.

Lid closure dynamics of porcine pancreatic lipase in aqueous solution.

BACKGROUND: Pancreatic lipases hydrolyze fatty acids in dietary pathway. The activity of porcine pancreatic lipase (PPL) is controlled by lid domain along with a coenzyme, colipase. The active open-state conformation of the protein could be induced by detergents or bile salts which would be further stabilized by binding of colipase. In the absence of these interactions, the lid preferably attains a closed conformation in water. METHODS: Molecular dynamic simulation was used to monitor the lid movement of PPL in open and closed conformations in water. Free energy surface was constructed from the simulation. Energy barriers and major structural changes during lid opening were evaluated. RESULTS: The lid closure of PPL in water from its open conformation might be initiated by columbic interactions which initially move the lid away from domain 1. This is followed by major dihedral changes on the lid residues which alter the trajectory of motion. The lid then swirls back towards domain 1 to attain closed conformation. This is accompanied with conformational changes around ß5- and ß9-loops as well. However, PPL in closed conformation shows only the domain movements and the lid remains in its closed conformation. CONCLUSIONS: PPL in closed conformation is stable in water and the open conformation is driven towards closed state. The lid follows a swirling trajectory during the closure. GENERAL SIGNIFICANCE: Conformational state of the lid regulates the activity and substrate specificity of PPL. Hence, it is essential to understand the lid dynamics and the role of specific amino acid residues involved.