Using AlphaFold2 and Molecular Dynamics Simulation to Model Protein Recognition.

In this chapter, we predict the structure of the Arabidopsis receptor-homology-transmembrane-RING-H2 isoform 1 (RMR1) in complex with the C-terminal sorting determinant of cruciferin (CRU1) by AlphaFold2 using the ColabFold web interface and to perform molecular dynamics simulation to probe the dynamics of the predicted structures. Our results predict that the C-terminal carboxylate group of ctVSD of CRU1 is recognized by the conserved Arg89 of the cargo-binding loop of RMR1 and Arg468 of CRU1 by negative charge residues in the cargo-binding pocket of RMR1. The procedures described here are useful for modeling of other protein complexes.

A spectrum of clinically-identified cysteine mutations in fibulin-3 (EFEMP1) highlight its disulfide bonding complexity and potential to induce stress response activation.

Fibulin-3 (FBLN3), also known as EFEMP1, is a secreted extracellular matrix (ECM) glycoprotein that contains forty cysteine residues. These cysteines, which are distributed across one atypical and five canonical calcium-binding epidermal growth factor (EGF) domains, are important for regulating FBLN3 structure, secretion, and presumably function. As evidence of this importance, a rare homozygous p.C55R mutation in FBLN3 negates its function, alters disulfide bonding, and causes marfanoid syndrome. Additional studies suggest that heterozygous premature stop codon mutations in FBLN3 may also cause similar, albeit less severe, connective tissue disorders. Interestingly, a series of twenty-four cysteine mutations in FBLN3 have been identified in the human population and published in the Clinical Variation (ClinVar) and gnomAD databases. We tested how seven of these cysteine mutants (five loss-of-cysteine variants: C42Y, C190R, C218R, C252F, and C365S, two gain-of-cysteine variants: R358C, Y369C) and two newly developed mutations (G57C and Y397C) altered FBLN3 secretion, disulfide bonding, MMP2 zymography, and stress response activation Surprisingly, we found a wide variety of biochemical behaviors: i) loss-of-cysteine variants correlated with an increased likelihood of disulfide dimer formation, ii) N-terminal mutations were less likely to disrupt secretion, and were less prone to aggregation, iii) in contrast to wild-type FBLN3, multiple, but not all variants failed to induce MMP2 levels in cell culture, and iv) C-terminal mutations (either loss or gain of cysteines) were more prone to significant secretion defects, intracellular accumulation/misfolding, and stress response activation. These results provide molecular and biochemical insight into FBLN3 folding, secretion, and function for many cysteine mutations found in the human population, some of which may increase the likelihood of subclinical connective tissue or other FBLN3-associated haploinsufficiency diseases.

Uncovering a Latent Bioactive Interleukin-6 Glycoform.

A bioinspired semisynthesis of human-interleukin-6 bearing N-glycan at Asn143 (143glycosyl-IL-6) was performed by intentional glycosylation effects and protein folding chemistry for regioselective peptide-backbone activation. 143Glycosyl-IL-6 is a genetically coded cytokine, but isolation was difficult owing to a tiny amount. IL6-polypeptide (1-141-position) with an intentionally inserted cysteine at 142-position was expressed in E. coli. The expressed polypeptide was treated with a chemical folding process to make a specific helices bundle conformation through native two-disulfide bonds (43-49 and 72-82). Utilizing the successfully formed free-142-cysteine, sequential conversions using cyanation of 142-cysteine, hydrazinolysis, and thioesterification created a long polypeptide (1-141)-thioester. However, the resultant polypeptide-thioester caused considerable aggregation owing to a highly hydrophobic peptide sequence. After the reduction of two-disulfide bonds of polypeptide (1-141)-thioester, an unprecedented hydrophilic N-glycan tag was inserted at the resultant cysteine thiols. The N-glycan tags greatly stabilized polypeptide-thioester. The subsequent native chemical ligation and desulfurization successfully gave a whole 143glycosyl-IL-6 polypeptide (183-amino acids). Removal of four N-glycan tags and immediate one-pot in vitro folding protocol efficiently produced the folded 143glycosyl-IL-6. The folded 143glycosyl-IL-6 exhibited potent cell proliferation activity. The combined studies with molecular dynamics simulation, semisynthesis, and bioassays predict the bioactive conformation of latent 143glycosyl-IL-6.

Origins of Life: The Protein Folding Problem all over again?

How did specific useful protein sequences arise from simpler molecules at the origin of life? This seemingly needle-in-a-haystack problem has remarkably close resemblance to the old Protein Folding Problem, for which the solution is now known from statistical physics. Based on the logic that Origins must have come only after there was an operative evolution mechanism-which selects on phenotype, not genotype-we give a perspective that proteins and their folding processes are likely to have been the primary driver of the early stages of the origin of life.

Insights into the interaction between UGGT, the gatekeeper of folding in the ER, and its partner, the selenoprotein SEP15.

The enzyme UDP-glucose: glycoprotein glucosyltransferase (UGGT) is the gatekeeper of protein folding within the endoplasmic reticulum (ER). One-third of the human proteome traverses the ER where folding and maturation are facilitated by a complex protein homeostasis network. Both glycan modifications and disulfide bonds are of key importance in the maturation of these ER proteins. The actions of UGGT are intimately linked to the glycan code for folding and maturation of secretory proteins in the ER. UGGT selectively glucosylates the N-linked glycan of misfolded proteins so that they can reenter the lectin-folding chaperone cycle and be retained within the ER for further attempts at folding. An intriguing aspect of UGGT function is its interaction with its poorly understood cochaperone, the 15 kDa selenoprotein known as SELENOF or SEP15. This small protein contains a rare selenocysteine residue proposed to act as an oxidoreductase toward UGGT substrates. AlphaFold2 predictions of the UGGT1/SEP15 complex provide insight into this complex at a structural level. The predicted UGGT1/SEP15 interaction interface was validated by mutagenesis and coimmunoprecipitation experiments. These results serve as a springboard for models of the integrated action of UGGT1 and SEP15.

Oscillatory hypoxia induced gene expression predicts low survival in human breast cancer patients.

Hypoxia is one of the key factors in the tumor microenvironment regulating nearly all steps in the metastatic cascade in many cancers, including in breast cancer. The hypoxic regions can however be dynamic with the availability of oxygen fluctuating or oscillating. The canonical response to hypoxia is relayed by transcription factor Hypoxia-Inducible Factor 1 (HIF-1), which is stabilized in hypoxia and acts as the master regulator of a large number of downstream genes. However, HIF-1 transcriptional activity can also fluctuate either due to unstable hypoxia, or by lactate mediated noncanonical degradation of HIF-1. Our understanding of how oscillatory hypoxia or HIF-1 activity specifically influences cancer malignancy is very limited. Here, using MDA-MB-231 cells as a model of triple negative breast cancer characterized by severe hypoxia, we measured the gene expression changes induced specifically by oscillatory hypoxia. We found that oscillatory hypoxia can specifically regulate gene expression differently, and at times opposite to stable hypoxia. Using the Cancer Genome Atlas RNAseq data of human cancer samples, we show that the oscillatory specific gene expression signature in MDA-MB-231 is enriched in most human cancers, and prognosticates low survival in breast cancer patients. In particular, we found that oscillatory hypoxia, unlike stable hypoxia, induces unfolded protein folding response in cells resulting in gene expression predicting reduced survival.

Using AlphaFold Multimer to discover interkingdom protein-protein interactions.

Structural prediction by artificial intelligence can be powerful new instruments to discover novel protein-protein interactions, but the community still grapples with the implementation, opportunities and limitations. Here, we discuss and re-analyse our in silico screen for novel pathogen-secreted inhibitors of immune hydrolases to illustrate the power and limitations of structural predictions. We discuss strategies of curating sequences, including controls, and reusing sequence alignments and highlight important limitations caused by different platforms, sequence depth and computing times. We hope these experiences will support similar interactomic screens by the research community.

Characterization of alternate encounter assemblies of SARS-CoV-2 main protease.

The assembly of two monomeric constructs spanning segments 1-199 (MPro1-199) and 10-306 (MPro10-306) of SARS-CoV-2 main protease (MPro) was examined to assess the existence of a transient heterodimer intermediate in the N-terminal autoprocessing pathway of MPro model precursor. Together, they form a heterodimer population accompanied by a 13-fold increase in catalytic activity. Addition of inhibitor GC373 to the proteins increases the activity further by ∼7-fold with a 1:1 complex and higher order assemblies approaching 1:2 and 2:2 molecules of MPro1-199 and MPro10-306 detectable by analytical ultracentrifugation and native mass estimation by light scattering. Assemblies larger than a heterodimer (1:1) are discussed in terms of alternate pathways of domain III association, either through switching the location of helix 201-214 onto a second helical domain of MPro10-306 and vice versa or direct interdomain III contacts like that of the native dimer, based on known structures and AlphaFold 3 prediction, respectively. At a constant concentration of MPro1-199 with molar excess of GC373, the rate of substrate hydrolysis displays first order dependency on the MPro10-306 concentration and vice versa. An equimolar composition of the two proteins with excess GC373 exhibits half-maximal activity at ∼6 µM MPro1-199. Catalytic activity arises primarily from MPro1-199 and is dependent on the interface interactions involving the N-finger residues 1-9 of MPro1-199 and E290 of MPro10-306. Importantly, our results confirm that a single N-finger region with its associated inter-subunit contacts is sufficient to form a heterodimeric MPro intermediate with enhanced catalytic activity.

Mechanochemical forces regulate the composition and function of CAT tails.

The ribosome-associated quality control (RQC) pathway resolves stalled ribosomes. As part of RQC, stalled nascent polypeptide chains (NCs) are appended with CArboxy-Terminal amino acids (CAT tails) in an mRNA-free, non-canonical elongation process. CAT tail composition includes Ala, Thr, and potentially other residues. The relationship between CAT tail composition and function has remained unknown. Using biochemical approaches in yeast, we discovered that mechanochemical forces on the NC regulate CAT tailing. We propose CAT tailing initially operates in an "extrusion mode" that increases NC lysine accessibility for on-ribosome ubiquitination. Thr in CAT tails enhances NC extrusion by preventing formation of polyalanine, which can form α-helices. After NC ubiquitylation, pulling forces on the NC switch CAT tailing to an Ala-only "release mode" which facilitates nascent chain release from large ribosomal subunits and NC degradation. Failure to switch from extrusion to release mode leads to accumulation of NCs on large ribosomal subunits and proteotoxic aggregation of Thr-rich CAT tails.

The importance of the location of the N-terminus in successful protein folding in vivo and in vitro.

Protein folding in the cell often begins during translation. Many proteins fold more efficiently cotranslationally than when refolding from a denatured state. Changing the vectorial synthesis of the polypeptide chain through circular permutation could impact functional, soluble protein expression and interactions with cellular proteostasis factors. Here, we measure the solubility and function of every possible circular permutant (CP) of HaloTag in Escherichia coli cell lysate using a gel-based assay, and in living E. coli cells via FACS-seq. We find that 78% of HaloTag CPs retain protein function, though a subset of these proteins are also highly aggregation-prone. We examine the function of each CP in E. coli cells lacking the cotranslational chaperone trigger factor and the intracellular protease Lon and find no significant changes in function as a result of modifying the cellular proteostasis network. Finally, we biophysically characterize two topologically interesting CPs in vitro via circular dichroism and hydrogen-deuterium exchange coupled with mass spectrometry to reveal changes in global stability and folding kinetics with circular permutation. For CP33, we identify a change in the refolding intermediate as compared to wild-type (WT) HaloTag. Finally, we show that the strongest predictor of aggregation-prone expression in cells is the introduction of termini within the refolding intermediate. These results, in addition to our finding that termini insertion within the conformationally restrained core is most disruptive to protein function, indicate that successful folding of circular permutants may depend more on changes in folding pathway and termini insertion in flexible regions than on the availability of proteostasis factors.

Evidence that the SARS-CoV-2 S protein undergoes a conformational change at the Golgi complex that leads to the formation of virus neutralising antibody binding epitopes in the S1 protein subunit.

Recombinant SARS-CoV-2 S protein expression was examined in Vero cells by imaging using the human monoclonal antibody panel (PD4, PD5, sc23, and sc29). The PD4 and sc29 antibodies recognised conformational specific epitopes in the S2 protein subunit at the Endoplasmic reticulum and Golgi complex. While PD5 and sc23 detected conformationally specific epitopes in the S1 protein subunit at the Golgi complex, only PD5 recognised the receptor binding domain (RBD). A comparison of the staining patterns of PD5 with non-conformationally specific antibodies that recognises the S1 subunit and RBD suggested the PD5 recognised a conformational structure within the S1 protein subunit. Our data suggests the antibody binding epitopes recognised by the human monoclonal antibodies formed at different locations in the secretory pathway during S protein transport, but a conformational change in the S1 protein subunit at the Golgi complex formed antibody binding epitopes that are recognised by virus neutralising antibodies.

Study of changes in folding/unfolding properties and stability of Arabidopsis thaliana MYB12 transcription factor following UV-B exposure in vitro.

Flavonoids mostly protect plant cells from the harmful effects of UV-B radiation from the sun. In plants, the R2R3-subfamily of the MYB transcription factor, MYB12, is a key inducer of the biosynthesis of flavonoids. Our study involves the biophysical characterization of Arabidopsis thaliana MYB12 protein (AtMYB12) under UV-B exposure in vitro. Tryptophan fluorescence studies using recombinant full-length AtMYB12 (native) and the N-terminal truncated versions (first N-terminal MYB domain absent in AtMYB12Δ1, and both the first and second N-terminal MYB domains absent in AtMYB12Δ2) have revealed prominent alteration in the tryptophan microenvironment in AtMYB12Δ1 and AtMYB12Δ2 protein as a result of UV-B exposure as compared with the native AtMYB12. Bis-ANS binding assay and urea-mediated denaturation profiling showed an appreciable change in the structural conformation in AtMYB12Δ1 and AtMYB12Δ2 proteins as compared with the native AtMYB12 protein following UV-B irradiation. UV-B-treated AtMYB12Δ2 showed a higher predisposition of aggregate formation in vitro. CD spectral analyses revealed a decrease in α-helix percentage with a concomitant increase in random coiled structure formation in AtMYB12Δ1 and AtMYB12Δ2 as compared to native AtMYB12 following UV-B treatment. Overall, these findings highlight the critical function of the N-terminal MYB domains in maintaining the stability and structural conformation of the AtMYB12 protein under UV-B stress in vitro.

The role of interleukin-10 in mitigating endoplasmic reticulum stress in aged mice through exercise.

While unfolded protein response (UPR) is essential for cellular protection, its prolonged activation may induce apoptosis, compromising cellular longevity. The aging process increases the endoplasmic reticulum (ER) stress in skeletal muscle. However, whether combined exercise can prevent age-induced ER stress in skeletal muscle remains unknown. Evidence suggests that ER stress may increase inflammation by counteracting the positive effects of interleukin-10 (IL-10), while its administration in cells inhibits ER stress and apoptosis. This study verified the effects of aging and combined exercise on physical performance, ER stress markers, and inflammation in the quadriceps of mice. Moreover, we verified the effects of IL-10 on ER stress markers. C57BL/6 mice were distributed into young (Y, 6-month-old), old sedentary (OS, sedentary, 24-month-old), and old trained group (OT, submitted to short-term combined exercise, 24-month-old). To clarify the role of IL-10 in UPR pathways, knockout mice lacking IL-10 were used. The OS mice presented worse physical performance and higher ER stress-related proteins, such as CHOP and p-eIF2α/eIF2α. The exercise protocol increased muscle strength and IL-10 protein levels in OT while inducing the downregulation of CHOP protein levels compared to OS. Furthermore, mice lacking IL-10 increased BiP, CHOP, and p-eIF2α/eIF2α protein levels, indicating this cytokine can regulate the ER stress response in skeletal muscle. Bioinformatics analysis showed that endurance and resistance training downregulated DDIT3 and XBP1 gene expression in the vastus lateralis of older people, reinforcing our findings. Thus, combined exercise is a potential therapeutic intervention for promoting adjustments in ER stress markers in aged skeletal muscle.

Divergent folding-mediated epistasis among unstable membrane protein variants.

Many membrane proteins are prone to misfolding, which compromises their functional expression at the plasma membrane. This is particularly true for the mammalian gonadotropin-releasing hormone receptor GPCRs (GnRHR). We recently demonstrated that evolutionary GnRHR modifications appear to have coincided with adaptive changes in cotranslational folding efficiency. Though protein stability is known to shape evolution, it is unclear how cotranslational folding constraints modulate the synergistic, epistatic interactions between mutations. We therefore compared the pairwise interactions formed by mutations that disrupt the membrane topology (V276T) or tertiary structure (W107A) of GnRHR. Using deep mutational scanning, we evaluated how the plasma membrane expression of these variants is modified by hundreds of secondary mutations. An analysis of 251 mutants in three genetic backgrounds reveals that V276T and W107A form distinct epistatic interactions that depend on both the severity and the mechanism of destabilization. V276T forms predominantly negative epistatic interactions with destabilizing mutations in soluble loops. In contrast, W107A forms positive interactions with mutations in both loops and transmembrane domains that reflect the diminishing impacts of the destabilizing mutations in variants that are already unstable. These findings reveal how epistasis is remodeled by conformational defects in membrane proteins and in unstable proteins more generally.

Reassessing the exon-foldon correspondence using frustration analysis.

Protein folding and evolution are intimately linked phenomena. Here, we revisit the concept of exons as potential protein folding modules across a set of 38 abundant and conserved protein families. Taking advantage of genomic exon-intron organization and extensive protein sequence data, we explore exon boundary conservation and assess the foldon-like behavior of exons using energy landscape theoretic measurements. We found deviations in the exon size distribution from exponential decay indicating selection in evolution. We show that when taken together there is a pronounced tendency to independent foldability for segments corresponding to the more conserved exons, supporting the idea of exon-foldon correspondence. While 45% of the families follow this general trend when analyzed individually, there are some families for which other stronger functional determinants, such as preserving frustrated active sites, may be acting. We further develop a systematic partitioning of protein domains using exon boundary hotspots, showing that minimal common exons correspond with uninterrupted alpha and/or beta elements for the majority of the families but not for all of them.

HOP stabilizes the HSFA1a and plays a main role in the onset of thermomorphogenesis.

Living organisms have the capacity to respond to environmental stimuli, including warm conditions. Upon sensing mild temperature, plants launch a transcriptional response that promotes morphological changes, globally known as thermomorphogenesis. This response is orchestrated by different hormonal networks and by the activity of different transcription factors, including the heat shock factor A1 (HSFA1) family. Members of this family interact with heat shock protein 70 (HSP70) and heat shock protein 90 (HSP90); however, the effect of this binding on the regulation of HSFA1 activity or of the role of cochaperones, such as the HSP70-HSP90 organizing protein (HOP) on HSFA1 regulation, remains unknown. Here, we show that AtHOPs are involved in the folding and stabilization of the HSFA1a and are required for the onset of the transcriptional response associated to thermomorphogenesis. Our results demonstrate that the three members of the AtHOP family bind in vivo to the HSFA1a and that the expression of multiple HSFA1a-responsive-responsive genes is altered in the hop1 hop2 hop3 mutant under warm temperature. Interestingly, HSFA1a is accumulated at lower levels in the hop1 hop2 hop3 mutant, while control levels are recovered in the presence of the proteasome inhibitor MG132 or the synthetic chaperone tauroursodeoxycholic acid (TUDCA). This uncovers the HSFA1a as a client of HOP complexes in plants and reveals the participation of HOPs in HSFA1a stability.

The Temperature Dependence of Hydrogen Bonds Is More Uniform in Stable Proteins: An Analysis of NMR h3JNC' Couplings in Four Different Protein Structures.

Long-range HNCO NMR spectra for proteins show crosspeaks due to 1JNC', 2JNC', 3JNCγ, and h3JNC' couplings. The h3JNC' couplings are transmitted through hydrogen bonds and their sizes are correlated to hydrogen bond lengths. We collected long-range HNCO data at a series of temperatures for four protein structures. P22i and CUS-3i are six-stranded beta-barrel I-domains from phages P22 and CUS-3 that share less than 40% sequence identity. The cis and trans states of the C-terminal domain from pore-forming toxin hemolysin ΙΙ (HlyIIC) arise from the isomerization of a single G404-P405 peptide bond. For P22i and CUS-3i, hydrogen bonds detected by NMR agree with those observed in the corresponding domains from cryoEM structures of the two phages. Hydrogen bond lengths derived from the h3JNC' couplings, however, are poorly conserved between the distantly related CUS-3i and P22i domains and show differences even between the closely related cis and trans state structures of HlyIIC. This is consistent with hydrogen bond lengths being determined by local differences in structure rather than the overall folding topology. With increasing temperature, hydrogen bonds typically show an apparent increase in length that has been attributed to protein thermal expansion. Some hydrogen bonds are invariant with temperature, however, while others show apparent decreases in length, suggesting they become stabilized with increasing temperature. Considering the data for the three proteins in this study and previously published data for ubiquitin and GB3, lowered protein folding stability and cooperativity corresponds with a larger range of temperature responses for hydrogen bonds. This suggests a partial uncoupling of hydrogen bond energetics from global unfolding cooperativity as protein stability decreases.

Producing recombinant proteins in Vibrio natriegens.

The diversity of chemical and structural attributes of proteins makes it inherently difficult to produce a wide range of proteins in a single recombinant protein production system. The nature of the target proteins themselves, along with cost, ease of use, and speed, are typically cited as major factors to consider in production. Despite a wide variety of alternative expression systems, most recombinant proteins for research and therapeutics are produced in a limited number of systems: Escherichia coli, yeast, insect cells, and the mammalian cell lines HEK293 and CHO. Recent interest in Vibrio natriegens as a new bacterial recombinant protein expression host is due in part to its short doubling time of ≤ 10 min but also stems from the promise of compatibility with techniques and genetic systems developed for E. coli. We successfully incorporated V. natriegens as an additional bacterial expression system for recombinant protein production and report improvements to published protocols as well as new protocols that expand the versatility of the system. While not all proteins benefit from production in V. natriegens, we successfully produced several proteins that were difficult or impossible to produce in E. coli. We also show that in some cases, the increased yield is due to higher levels of properly folded protein. Additionally, we were able to adapt our enhanced isotope incorporation methods for use with V. natriegens. Taken together, these observations and improvements allowed production of proteins for structural biology, biochemistry, assay development, and structure-based drug design in V. natriegens that were impossible and/or unaffordable to produce in E. coli.

Synonymous and non-synonymous codon substitutions can alleviate dependence on GroEL for folding.

The Escherichia coli GroEL/ES chaperonin system facilitates protein folding in an ATP-driven manner. There are <100 obligate clients of this system in E. coli although GroEL can interact and assist the folding of a multitude of proteins in vitro. It has remained unclear, however, which features distinguish obligate clients from all the other proteins in an E. coli cell. To address this question, we established a system for selecting mutations in mouse dihydrofolate reductase (mDHFR), a GroEL interactor, that diminish its dependence on GroEL for folding. Strikingly, both synonymous and non-synonymous codon substitutions were found to reduce mDHFR's dependence on GroEL. The non-synonymous substitutions increase the rate of spontaneous folding whereas computational analysis indicates that the synonymous substitutions appear to affect translation rates at specific sites.