Lignocellulose is the most abundant natural biopolymer on earth and a potential raw material for the production of fuels and chemicals. However, only some organisms such as bacteria and fungi produce enzymes that metabolize this polymer. In this work we have demonstrated the presence of cellulolytic activity in the supernatant of Scenedesmus quadricauda cultures and we identified the presence of extracellular cellulases in the genome of five Scenedesmus species. Scenedesmus is a green alga which grows in both freshwater and saltwater regions as well as in soils, showing highly flexible metabolic properties. Sequence comparison of the different identified cellulases with hydrolytic enzymes from other organisms using multisequence alignments and phylogenetic trees showed that these proteins belong to the families of glycosyl hydrolases 1, 5, 9, and 10. In addition, most of the Scenedesmus cellulases showed greater sequence similarity with those from invertebrates, fungi, bacteria, and other microalgae than with the plant homologs. Furthermore, the data obtained from the three dimensional structure showed that both, their global structure and the main amino acid residues involved in catalysis and substrate binding are well conserved. Based on our results, we propose that different species of Scenedesmus could act as biocatalysts for the hydrolysis of cellulosic biomass produced from sunlight.

Many human diseases are associated with the misfolding of amyloidogenic proteins. Understanding the mechanisms cells employ to ensure the integrity of the proteome is therefore a crucial step in the development of potential therapeutic interventions. Yeast cells possess numerous prion-forming proteins capable of adopting amyloid conformations, possibly as an epigenetic mechanism to cope with changing environmental conditions. The ribosome-associated complex (RAC), which docks near the ribosomal polypeptide exit tunnel and recruits the Hsp70 Ssb to chaperone nascent chains, can moderate the acquisition of these amyloid conformations in yeast. Here we examine the ability of the human RAC chaperone proteins Mpp11 and Hsp70L1 to function in place of their yeast RAC orthologues Zuo1 and Ssz1 in yeast lacking endogenous RAC and investigate the extent to which the human orthologues can perform RAC chaperone activities in yeast. We found that the Mpp11/Hsp70L1 complex can partially correct the growth defect seen in RAC-deficient yeast cells, although yeast/human hetero species complexes were variable in this ability. The proportion of cells in which the Sup35 protein undergoes spontaneous conversion to a [PSI+ ] prion conformation, which is increased in the absence of RAC, was reduced by the presence of the human RAC complex. However, the toxicity in yeast from expression of a pathogenically expanded polyQ protein was unable to be countered by the human RAC chaperones. This yeast system can serve as a facile model for studying the extent to which the human RAC chaperones contribute to combating cotranslational misfolding of other mammalian disease-associated proteins.

The study aimed to screen prospective molecular targets of BCC and potential natural lead candidates as effective binders by computational modeling, molecular docking, and dynamic (MD) simulation studies. Based on the virulent functions, tRNA 5-methylaminomethyl-2-thiouridine biosynthesis bifunctional protein (mnmC) and pyrimidine/purine nucleoside phosphorylase (ppnP) were selected as the prospective molecular targets. In the absence of experimental data, the three-dimensional (3D) structures of these targets were computationally predicted. After a thorough literature survey and database search, the drug-likeness, and pharmacokinetic properties of 70 natural molecules were computationally predicted and the effectual binding of the best lead molecules against both the targets was predicted by molecular docking. The stabilities of the best-docked complexes were validated by MD simulation and the binding energy calculations were carried out by MM-GBSA approaches. The present study revealed that the hypothetical models of mnmC and ppnP showed stereochemical accuracy. The study also showed that among 70 natural compounds subjected to computational screening, Honokiol (3',5-Di(prop-2-en-1-yl) [1,1'-biphenyl]-2,4'-diol) present in Magnolia showed ideal drug-likeness, pharmacokinetic features and showed effectual binding with mnmC and ppnP (binding energies -7.3 kcal/mol and -6.6 kcal/mol, respectively). The MD simulation and GBSA calculation studies showed that the ligand-protein complexes stabilized throughout tMD simulation. The present study suggests that Honokiol can be used as a potential lead molecule against mnmC and ppnP targets of BCC and this study provides insight into further experimental validation for alternative lead development against drug resistant BCC.

AlphaFold2 has revolutionized protein structure prediction from amino-acid sequence. In addition to protein structures, high-resolution dynamics information about various protein regions is important for understanding protein function. Although AlphaFold2 has neither been designed nor trained to predict protein dynamics, it is shown here how the information returned by AlphaFold2 can be used to predict dynamic protein regions at the individual residue level. The approach, which is termed cdsAF2, uses the 3D protein structure returned by AlphaFold2 to predict backbone NMR NH S2 order parameters using a local contact model that takes into account the contacts made by each peptide plane along the backbone with its environment. By combining for each residue AlphaFold2's pLDDT confidence score for the structure prediction accuracy with the predicted S2 value using the local contact model, an estimator is obtained that semi-quantitatively captures many of the dynamics features observed in experimental backbone NMR NH S2 order parameter profiles. The method is demonstrated for a set nine proteins of different sizes and variable amounts of dynamics and disorder.

The AMP-activated protein kinase (AMPK) is known to be activated by the protein tyrosine phosphatase non-receptor type 12 (PTP-PEST) under hypoxic conditions. This activation is mediated by tyrosine dephosphorylation of the AMPKα subunit. However, the identity of the phosphotyrosine residues that PTP-PEST dephosphorylates remains unknown. In this study, we first predicted the structure of the complex of the AMPKα2 subunit and PTP-PEST catalytic domain using bioinformatics tools and further confirmed the stability of the complex using molecular dynamics simulations. Evaluation of the protein-protein interfaces indicated that residue Tyr232 is the most likely dephosphorylation site on AMPKα2. In addition, we explored the effect of phosphorylation of PTP-PEST residue Tyr64 on the stability of the complex. Phosphorylation of the highly conserved Tyr64, an interface residue, enhances the stability of the complex via the rearrangement of a network of electrostatic interactions in conjunction with conformational changes in the catalytic WPD loop. We generated a phosphomimetic (PTP-PEST-Y64D) mutant and used co-immunoprecipitation to study the effect of PTP-PEST phosphorylation on AMPKα2 binding. The mutant exhibited an increased affinity for AMPKα2 and corroborated the in-silico predictions. Together, our findings present a plausible structural basis of AMPK regulation by PTP-PEST and show how phosphorylation of PTP-PEST affects its interaction with AMPKα2.

Human carnosinases (CNs) are dimeric dipeptidases in the metallopeptidase M20 family. Two isoforms of carnosinases (Zn2+ -containing carnosinase 1 (CN1) found in serum and Mn2+ -carnosinase 2 (CN2) in tissue) were identified. Both CNs cleave histidine-containing (Xaa-His) dipeptides such as carnosine where CN2 was found to accept a broader spectrum of substrates. A loss of CN function, resulting in a high carnosine concentration, reduces risk for diabetes and neurological disorders. Although several studies on CN activities and its Michaelis complex were conducted, all shed the light on CN1 activity where the CN2 data is limited. Also, the molecular details on CN1 and CN2 similarity and dissimilarity in structure and function remain unclear. Thus, in this work, molecular dynamics (MD) simulations were employed to study structure and dynamics of human CN1 and CN2 in comparison. The results show that the different catalytic ability of both CNs is due to their pocket size and environment. CN2 can accept a wider range of substrate due to the wider mouth of a binding pocket. The L1 loop seems to play a role in gating activity. Comparing to CN2, CN1 provides more electronegative entrance, more wettability, and higher stability of catalytic metal ion-pair in the active site which allow more efficient water-mediated catalysis. The microscopic understanding obtained here can serve as a basis for CN inhibition strategies resulting in higher carnosine levels and consequently mitigating complications associated with diseases such as diabetes and neurological disorder.

The microbial enzyme DapE plays a critical role in the lysine biosynthetic pathway and is considered as a potentially safe antibiotic target. In this study, atomistic simulations are employed to identify the modes of essential dynamics that define the conformational response of the enzyme to ligand binding and unbinding. The binding modes and the binding affinities of the products to the DapE enzyme are estimated from the MM-PBSA method, and the residues contributing to the ligand binding are identified. Various structural analyses and the principal component analysis of the molecular dynamics trajectories reveal that the removal of products from the active site causes a significant change in the overall enzyme structure. Both Cartesian and dihedral principal component analyses are used to characterize the structural changes in terms of domain unfolding and domain twisting motions. In the most dominant mode, that is, the domain unfolding motion, the two catalytic domains move away from the two dimerization domains of the dimeric enzyme, representing a closed-to-open conformational change. The conformational changes are initiated by the coordinated movement of three loops (Asp75-Pro82, Gly240-Asn244, and Thr347-Glu353) that trigger a domain-level movement. From multiple short trajectories, the time constant associated with the domain opening motion is estimated as 43.6 ns. Physiologically, this close-to-open conformational change is essential for the regeneration of the initial state of the enzyme for the subsequent cycle of catalytic action and provides the apo enzyme enough flexibility for efficient substrate binding.

Degradation of solid polyethylene terephthalate (PET) by leaf branch compost cutinase (LCC) produces various PET-derived degradation intermediates (DIs), in addition to terephthalic acid (TPA), which is the recyclable terminal product of all PET degradation. Although DIs can also be converted into TPA, in solution, by LCC, the TPA that is obtained through enzymatic degradation of PET, in practice, is always contaminated by DIs. Here, we demonstrate that the primary reason for non-degradation of DIs into TPA in solution is the efficient binding of LCC onto the surface of solid PET. Although such binding enhances the degradation of solid PET, it depletes the surrounding solution of enzyme that could otherwise have converted DIs into TPA. To retain a subpopulation of enzyme in solution that would mainly degrade DIs, we introduced mutations to reduce the hydrophobicity of areas surrounding LCC's active site, with the express intention of reducing LCC's binding to solid PET. Despite the consequent reduction in invasion and degradation of solid PET, overall levels of production of TPA were ~3.6-fold higher, due to the partitioning of enzyme between solid PET and the surrounding solution, and the consequent heightened production of TPA from DIs. Further, synergy between such mutated LCC (F125L/F243I LCC) and wild-type LCC resulted in even higher yields, and TPA of nearly ~100% purity.

Multiple severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants continue to evolve carrying flexible amino acid substitutions in the spike protein's receptor binding domain (RBD). These substitutions modify the binding of the SARS-CoV-2 to human angiotensin-converting enzyme 2 (hACE2) receptor and have been implicated in altered host fitness, transmissibility, and efficacy against antibody therapeutics and vaccines. Reliably predicting the binding strength of SARS-CoV-2 variants RBD to hACE2 receptor and neutralizing antibodies (NAbs) can help assessing their fitness, and rapid deployment of effective antibody therapeutics, respectively. Here, we introduced a two-step computational framework with 3-fold validation that first identified dissociation constant as a reliable predictor of binding affinity in hetero- dimeric and trimeric protein complexes. The second step implements dissociation constant as descriptor of the binding strengths of SARS-CoV-2 variants RBD to hACE2 and NAbs. Then, we examined several variants of concerns (VOCs) such as Alpha, Beta, Gamma, Delta, and Omicron and demonstrated that these VOCs RBD bind to the hACE2 with enhanced affinity. Furthermore, the binding affinity of Omicron variant's RBD was reduced with majority of the RBD-directed NAbs, which is highly consistent with the experimental neutralization data. By studying the atomic contacts between RBD and NAbs, we revealed the molecular footprints of four NAbs (GH-12, P2B-1A1, Asarnow_3D11, and C118)-that may likely neutralize the recently emerged Omicron variant-facilitating enhanced binding affinity. Finally, our findings suggest a computational pathway that could aid researchers identify a range of current NAbs that may be effective against emerging SARS-CoV-2 variants.

Inactive rhodopsin can absorb photons, which induces different structural transitions that finally activate rhodopsin. We have examined the change in spatial configurations and physicochemical factors that result during the transition mechanism from the inactive to the active rhodopsin state via intermediates. During the activation process, many existing atomic contacts are disrupted, and new ones are formed. This is related to the movement of Helix 5, which tilts away from Helix 3 in the intermediate state in lumirhodopsin and moves closer to Helix 3 again in the active state. Similar patterns of changing atomic contacts are observed between Helices 3 and 5 of the adenosine and neurotensin receptors. In addition, residues 220-238 of rhodopsin, which are disordered in the inactive state, fold in the active state before binding to the Gα, where it catalyzes GDP/GTP exchange on the Gα subunit. Finally, molecular dynamics simulations in the membrane environment revealed that the arrestin binding region adopts a more flexible extended conformation upon phosphorylation, likely promoting arrestin binding and inactivation. In summary, our results provide additional structural understanding of specific rhodopsin activation which might be relevant to other Class A G protein-coupled receptor proteins.

The G66V pathological variant of the coiled-coil-helix-coiled-coil-helix domain-containing protein 10 (CHCHD10), mitochondrial, plays a role in Jokela type spinal muscular atrophy. The wild-type and G66V mutant-type CHCHD10 proteins contain intrinsically disordered regions, and therefore, their structural ensemble studies have been experiencing difficulties using conventional tools. Here, we show our results regarding the first characterization of the structural ensemble characteristics of the G66V mutant form of CHCHD10 and the first comparison of these characteristics with the structural ensemble properties of wild-type CHCHD10. We find that the structural properties, potential of mean force surfaces, and principal component analysis show stark differences between these two proteins. These results are important for a better pathology, biochemistry and structural biology understanding of CHCHD10 and its G66V genetic variant and it is likely that these reported structural properties are important for designing more efficient treatments for the Jokela type of spinal muscular atrophy disease.