Effects of Flavourzyme and Alkaline Protease Treatment on Structure and Allergenicity of Peanut Allergen Ara h 1.

Research background: Peanut allergy poses a significant threat to human health due to the increased risk of long-term morbidity at low doses. Modifying protein structure to affect sensitization is a popular topic. Experimental approach: In this study, the purified peanut allergen Ara h 1 was enzymatically hydrolysed using Flavourzyme, alkaline protease or a combination of both. The binding ability of Ara h 1 to antibodies, gene expression and secretion levels of the proinflammatory factors interleukin-5 and interleukin-6 in Caco-2 cells was measured. Changes in the secondary and tertiary structures before and after treatment with Ara h 1 were analysed by circular dichroism and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Results and conclusions: The results indicated a decrease of the allergenicity and proinflammatory ability of Ara h 1. The evaluation showed that the Flavourzyme and alkaline protease treatments caused particle shortening and aggregation. The fluorescence emission peak increased by 3.4-fold after the combined treatment with both proteases. Additionally, the secondary structure underwent changes and the hydrophobicity also increased 8.95-fold after the combined treatment. Novelty and scientific contribution: These findings partially uncover the mechanism of peanut sensitization and provide an effective theoretical basis for the development of a new method of peanut desensitization.

Orthology-based analysis helps map evolutionary diversification and predict substrate class use of BAHD acyltransferases.

Large enzyme families catalyze metabolic diversification by virtue of their ability to use diverse chemical scaffolds. How enzyme families attain such functional diversity is not clear. Furthermore, duplication and promiscuity in such enzyme families limits their functional prediction, which has produced a burgeoning set of incompletely annotated genes in plant genomes. Here, we address these challenges using BAHD acyltransferases as a model. This fast-evolving family expanded drastically in land plants, increasing from one to five copies in algae to approximately 100 copies in diploid angiosperm genomes. Compilation of >160 published activities helped visualize the chemical space occupied by this family and define eight different classes based on structural similarities between acceptor substrates. Using orthologous groups (OGs) across 52 sequenced plant genomes, we developed a method to predict BAHD acceptor substrate class utilization as well as origins of individual BAHD OGs in plant evolution. This method was validated using six novel and 28 previously characterized enzymes and helped improve putative substrate class predictions for BAHDs in the tomato genome. Our results also revealed that while cuticular wax and lignin biosynthetic activities were more ancient, anthocyanin acylation activity was fixed in BAHDs later near the origin of angiosperms. The OG-based analysis enabled identification of signature motifs in anthocyanin-acylating BAHDs, whose importance was validated via molecular dynamic simulations, site-directed mutagenesis and kinetic assays. Our results not only describe how BAHDs contributed to evolution of multiple chemical phenotypes in the plant world but also propose a biocuration-enabled approach for improved functional annotation of plant enzyme families.

Insight into de-regulation of amino acid feedback inhibition: a focus on structure analysis method.

Regulation of amino acid's biosynthetic pathway is of significant importance to maintain homeostasis and cell functions. Amino acids regulate their biosynthetic pathway by end-product feedback inhibition of enzymes catalyzing committed steps of a pathway. Discovery of new feedback resistant enzyme variants to enhance industrial production of amino acids is a key objective in industrial biotechnology. Deregulation of feedback inhibition has been achieved for various enzymes using in vitro and in silico mutagenesis techniques. As enzyme's function, its substrate binding capacity, catalysis activity, regulation and stability are dependent on its structural characteristics, here, we provide detailed structural analysis of all feedback sensitive enzyme targets in amino acid biosynthetic pathways. Current review summarizes information regarding structural characteristics of various enzyme targets and effect of mutations on their structures and functions especially in terms of deregulation of feedback inhibition. Furthermore, applicability of various experimental as well as computational mutagenesis techniques to accomplish feedback resistance has also been discussed in detail to have an insight into various aspects of research work reported in this particular field of study.

DeepSTABp: A Deep Learning Approach for the Prediction of Thermal Protein Stability.

Proteins are essential macromolecules that carry out a plethora of biological functions. The thermal stability of proteins is an important property that affects their function and determines their suitability for various applications. However, current experimental approaches, primarily thermal proteome profiling, are expensive, labor-intensive, and have limited proteome and species coverage. To close the gap between available experimental data and sequence information, a novel protein thermal stability predictor called DeepSTABp has been developed. DeepSTABp uses a transformer-based protein language model for sequence embedding and state-of-the-art feature extraction in combination with other deep learning techniques for end-to-end protein melting temperature prediction. DeepSTABp can predict the thermal stability of a wide range of proteins, making it a powerful and efficient tool for large-scale prediction. The model captures the structural and biological properties that impact protein stability, and it allows for the identification of the structural features that contribute to protein stability. DeepSTABp is available to the public via a user-friendly web interface, making it accessible to researchers in various fields.

Optimization and validation of multi-state NMR protein structures using structural correlations.

Recent advances in the field of protein structure determination using liquid-state NMR enable the elucidation of multi-state protein conformations that can provide insight into correlated and non-correlated protein dynamics at atomic resolution. So far, NMR-derived multi-state structures were typically evaluated by means of visual inspection of structure superpositions, target function values that quantify the violation of experimented restraints and root-mean-square deviations that quantify similarity between conformers. As an alternative or complementary approach, we present here the use of a recently introduced structural correlation measure, PDBcor, that quantifies the clustering of protein states as an additional measure for multi-state protein structure analysis. It can be used for various assays including the validation of experimental distance restraints, optimization of the number of protein states, estimation of protein state populations, identification of key distance restraints, NOE network analysis and semiquantitative analysis of the protein correlation network. We present applications for the final quality analysis stages of typical multi-state protein structure calculations.

Resolving the zinc binding capacity of honey bee vitellogenin and locating its putative binding sites.

The protein vitellogenin (Vg) plays a central role in lipid transportation in most egg-laying animals. High Vg levels correlate with stress resistance and lifespan potential in honey bees (Apis mellifera). Vg is the primary circulating zinc-carrying protein in honey bees. Zinc is an essential metal ion in numerous biological processes, including the function and structure of many proteins. Measurements of Zn2+ suggest a variable number of ions per Vg molecule in different animal species, but the molecular implications of zinc-binding by this protein are not well-understood. We used inductively coupled plasma mass spectrometry to determine that, on average, each honey bee Vg molecule binds 3 Zn2+ -ions. Our full-length protein structure and sequence analysis revealed seven potential zinc-binding sites. These are located in the ß-barrel and α-helical subdomains of the N-terminal domain, the lipid binding site, and the cysteine-rich C-terminal region of unknown function. Interestingly, two potential zinc-binding sites in the ß-barrel can support a proposed role for this structure in DNA-binding. Overall, our findings suggest that honey bee Vg bind zinc at several functional regions, indicating that Zn2+ -ions are important for many of the activities of this protein. In addition to being potentially relevant for other egg-laying species, these insights provide a platform for studies of metal ions in bee health, which is of global interest due to recent declines in pollinator numbers.

Structural and functional characterization of ß2 -glycoprotein I domain 1 in anti-melanoma cell migration.

We previously found that circulating ß2 -glycoprotein I inhibits human endothelial cell migration, proliferation, and angiogenesis by diverse mechanisms. In the present study, we investigated the antitumor activities of ß2 -glycoprotein I using structure-function analysis and mapped the critical region within the ß2 -glycoprotein I peptide sequence that mediates anticancer effects. We constructed recombinant cDNA and purified different ß2 -glycoprotein I polypeptide domains using a baculovirus expression system. We found that purified ß2 -glycoprotein I, as well as recombinant ß2 -glycoprotein I full-length (D12345), polypeptide domains I-IV (D1234), and polypeptide domain I (D1) significantly inhibited melanoma cell migration, proliferation and invasion. Western blot analyses were used to determine the dysregulated expression of proteins essential for intracellular signaling pathways in B16-F10 treated with ß2 -glycoprotein I and variant recombinant polypeptides. Using a melanoma mouse model, we found that D1 polypeptide showed stronger potency in suppressing tumor growth. Structural analysis showed that fragments A and B within domain I would be the critical regions responsible for antitumor activity. Annexin A2 was identified as the counterpart molecule for ß2 -glycoprotein I by immunofluorescence and coimmunoprecipitation assays. Interaction between specific amino acids of ß2 -glycoprotein I D1 and annexin A2 was later evaluated by the molecular docking approach. Moreover, five amino acid residues were selected from fragments A and B for functional evaluation using site-directed mutagenesis, and P11A, M42A, and I55P mutations were shown to disrupt the anti-melanoma cell migration ability of ß2 -glycoprotein I. This is the first study to show the therapeutic potential of ß2 -glycoprotein I D1 in the treatment of melanoma progression.

A novel single amino acid deletion impairs fibronectin function and causes familial glomerulopathy with fibronectin deposits: case report of a family.

BACKGROUND: Glomerulopathy with fibronectin deposits is an autosomal dominant disease associated with proteinuria, hematuria, hypertension and renal function decline. Forty percent of the cases are caused by mutations in FN1, the gene that encodes fibronectin. CASE PRESENTATION: This report describes two cases of Glomerulopathy with fibronectin deposits, involving a 47-year-old father and a 14-year-old son. The renal biopsies showed glomeruli with endocapillary hypercellularity and large amounts of mesangial and subendothelial eosinophilic deposits. Immunohistochemistry for fibronectin was markedly positive. Whole exome sequencing identified a novel FN1 mutation that leads to an amino-acid deletion in both patients (Ile1988del), a variant that required primary amino-acid sequence analysis for assessment of pathogenicity. Our primary sequence analyses revealed that Ile1988 is very highly conserved among relative sequences and is positioned in a C-terminal FN3 domain containing heparin- and fibulin-1-binding sites. This mutation was predicted as deleterious and molecular mechanics simulations support that it can change the tertiary structure and affect the complex folding and its molecular functionality. CONCLUSION: The current report not only documents the occurrence of two GFND cases in an affected family and deeply characterizes its anatomopathological features but also identifies a novel pathogenic mutation in FN1, analyzes its structural and functional implications, and supports its pathogenicity.

Finding High-Quality Metal Ion-Centric Regions Across the Worldwide Protein Data Bank.

As the number of macromolecular structures in the worldwide Protein Data Bank (wwPDB) continues to grow rapidly, more attention is being paid to the quality of its data, especially for use in aggregated structural and dynamics analyses. In this study, we systematically analyzed 3.5 Å regions around all metal ions across all PDB entries with supporting electron density maps available from the PDB in Europe. All resulting metal ion-centric regions were evaluated with respect to four quality-control criteria involving electron density resolution, atom occupancy, symmetry atom exclusion, and regional electron density discrepancy. The resulting list of metal binding sites passing all four criteria possess high regional structural quality and should be beneficial to a wide variety of downstream analyses. This study demonstrates an approach for the pan-PDB evaluation of metal binding site structural quality with respect to underlying X-ray crystallographic experimental data represented in the available electron density maps of proteins. For non-crystallographers in particular, we hope to change the focus and discussion of structural quality from a global evaluation to a regional evaluation, since all structural entries in the wwPDB appear to have both regions of high and low structural quality.

From Protein Features to Sensing Surfaces.

Proteins play a major role in biosensors in which they provide catalytic activity and specificity in molecular recognition. However, the immobilization process is far from straightforward as it often affects the protein functionality. Extensive interaction of the protein with the surface or significant surface crowding can lead to changes in the mobility and conformation of the protein structure. This review will provide insights as to how an analysis of the physico-chemical features of the protein surface before the immobilization process can help to identify the optimal immobilization approach. Such an analysis can help to preserve the functionality of the protein when on a biosensor surface.

Alpha shape and Delaunay triangulation in studies of protein-related interactions.

In recent years, more 3D protein structures have become available, which has made the analysis of large molecular structures much easier. There is a strong demand for geometric models for the study of protein-related interactions. Alpha shape and Delaunay triangulation are powerful tools to represent protein structures and have advantages in characterizing the surface curvature and atom contacts. This review presents state-of-the-art applications of alpha shape and Delaunay triangulation in the studies on protein-DNA, protein-protein, protein-ligand interactions and protein structure analysis.

Characterizing of functional human coding RNA editing from evolutionary, structural, and dynamic perspectives.

A-to-I RNA editing has been recently shown to be a widespread phenomenon with millions of sites spread in the human transcriptome. However, only few are known to be located in coding sequences and modify the amino acid sequence of the protein product. Here, we used high-throughput data, variant prediction tools, and protein structural information in order to find structural and functional preferences for coding RNA editing. We show that RNA editing has a unique pattern of amino acid changes characterized by enriched stop-to-tryptophan changes, positive-to-neutral and neutral-to-positive charge changes. RNA editing tends to have stronger structural effect than equivalent A-to-G SNPs but weaker effect than random A-to-G mutagenesis events. Sites edited at low level tend to be located at conserved positions with stronger predicted deleterious effect on proteins comparing to sites edited at high frequencies. Lowly edited sites tend to destabilize the protein structure and affect amino acids with larger number of intra-molecular contacts. Still, some highly edited sites are predicted also to prominently affect structure and tend to be located at critical positions of the protein matrix and are likely to be functionally important. Using our pipeline, we identify and discuss several novel putative functional coding changing editing sites in the genes COPA (I164V), GIPC1 (T62A), ZN358 (K382R), and CCNI (R75G).

SNP and Structural Study of the Notch Superfamily Provides Insights and Novel Pharmacological Targets against the CADASIL Syndrome and Neurodegenerative Diseases.

The evolutionary conserved Notch signaling pathway functions as a mediator of direct cell-cell communication between neighboring cells during development. Notch plays a crucial role in various fundamental biological processes in a wide range of tissues. Accordingly, the aberrant signaling of this pathway underlies multiple genetic pathologies such as developmental syndromes, congenital disorders, neurodegenerative diseases, and cancer. Over the last two decades, significant data have shown that the Notch signaling pathway displays a significant function in the mature brains of vertebrates and invertebrates beyond neuronal development and specification during embryonic development. Neuronal connection, synaptic plasticity, learning, and memory appear to be regulated by this pathway. Specific mutations in human Notch family proteins have been linked to several neurodegenerative diseases including Alzheimer's disease, CADASIL, and ischemic injury. Neurodegenerative diseases are incurable disorders of the central nervous system that cause the progressive degeneration and/or death of brain nerve cells, affecting both mental function and movement (ataxia). There is currently a lot of study being conducted to better understand the molecular mechanisms by which Notch plays an essential role in the mature brain. In this study, an in silico analysis of polymorphisms and mutations in human Notch family members that lead to neurodegenerative diseases was performed in order to investigate the correlations among Notch family proteins and neurodegenerative diseases. Particular emphasis was placed on the study of mutations in the Notch3 protein and the structure analysis of the mutant Notch3 protein that leads to the manifestation of the CADASIL syndrome in order to spot possible conserved mutations and interpret the effect of these mutations in the Notch3 protein structure. Conserved mutations of cysteine residues may be candidate pharmacological targets for the potential therapy of CADASIL syndrome.

Microplastics in freshwater food chains: Priority list based on identification of oxidative stress response characteristic.

Exogenous exposure to high concentrations of microplastics (MPs) cause oxidative damage to freshwater food chains (FFCs). Thus, the patterns and mechanisms of oxidative stress responses (OSRs) induced by MPs in FFC organisms were investigated using theoretical simulation methods. Results showed an increasing (reduced) OSR was found in lower trophic levels (higher trophic levels). Besides, polycarbonate (polyvinyl chloride) causes the most (least) significant OSRs in FFC organisms, respectively. The impacts of MP additives were also analyzed using the full factorial experimental design, revealing flame retardants significantly influence oxidative stress variability. A constructive solution of "restriction-control-focus" is proposed for different types of MPs by the coefficient of variation-corrected CRITIC and the nested mean classification method. The mechanism analysis revealed a positive correlation between protein secondary structure orderliness and OSRs. Proteins in organisms that contain a high proportion of hydrophobic non-polar amino acids are more likely to bind to MP and enhance OSRs. This is the first study assessing the OSR patterns and ecological risks of MPs and their additives in FFCs with a proposed priority list, providing theoretical support for risk assessments and management strategies in freshwater environments.

Exaptation of Inactivated Host Enzymes for Structural Roles in Orthopoxviruses and Novel Folds of Virus Proteins Revealed by Protein Structure Modeling.

Viruses with large, double-stranded DNA genomes captured the majority of their genes from their hosts at different stages of evolution. The origins of many virus genes are readily detected through significant sequence similarity with cellular homologs. In particular, this is the case for virus enzymes, such as DNA and RNA polymerases or nucleotide kinases, that retain their catalytic activity after capture by an ancestral virus. However, a large fraction of virus genes have no readily detectable cellular homologs, meaning that their origins remain enigmatic. We explored the potential origins of such proteins that are encoded in the genomes of orthopoxviruses, a thoroughly studied virus genus that includes major human pathogens. To this end, we used AlphaFold2 to predict the structures of all 214 proteins that are encoded by orthopoxviruses. Among the proteins of unknown provenance, structure prediction yielded clear indications of origin for 14 of them and validated several inferences that were previously made via sequence analysis. A notable emerging trend is the exaptation of enzymes from cellular organisms for nonenzymatic, structural roles in virus reproduction that is accompanied by the disruption of catalytic sites and by an overall drastic divergence that precludes homology detection at the sequence level. Among the 16 orthopoxvirus proteins that were found to be inactivated enzyme derivatives are the poxvirus replication processivity factor A20, which is an inactivated NAD-dependent DNA ligase; the major core protein A3, which is an inactivated deubiquitinase; F11, which is an inactivated prolyl hydroxylase; and more similar cases. For nearly one-third of the orthopoxvirus virion proteins, no significantly similar structures were identified, suggesting exaptation with subsequent major structural rearrangement that yielded unique protein folds. IMPORTANCE Protein structures are more strongly conserved in evolution than are amino acid sequences. Comparative structural analysis is particularly important for inferring the origins of viral proteins that typically evolve at high rates. We used a powerful protein structure modeling method, namely, AlphaFold2, to model the structures of all orthopoxvirus proteins and compared them to all available protein structures. Multiple cases of recruitment of host enzymes for structural roles in viruses, accompanied by the disruption of catalytic sites, were discovered. However, many viral proteins appear to have evolved unique structural folds.

COLLAPSE: A representation learning framework for identification and characterization of protein structural sites.

The identification and characterization of the structural sites which contribute to protein function are crucial for understanding biological mechanisms, evaluating disease risk, and developing targeted therapies. However, the quantity of known protein structures is rapidly outpacing our ability to functionally annotate them. Existing methods for function prediction either do not operate on local sites, suffer from high false positive or false negative rates, or require large site-specific training datasets, necessitating the development of new computational methods for annotating functional sites at scale. We present COLLAPSE (Compressed Latents Learned from Aligned Protein Structural Environments), a framework for learning deep representations of protein sites. COLLAPSE operates directly on the 3D positions of atoms surrounding a site and uses evolutionary relationships between homologous proteins as a self-supervision signal, enabling learned embeddings to implicitly capture structure-function relationships within each site. Our representations generalize across disparate tasks in a transfer learning context, achieving state-of-the-art performance on standardized benchmarks (protein-protein interactions and mutation stability) and on the prediction of functional sites from the Prosite database. We use COLLAPSE to search for similar sites across large protein datasets and to annotate proteins based on a database of known functional sites. These methods demonstrate that COLLAPSE is computationally efficient, tunable, and interpretable, providing a general-purpose platform for computational protein analysis.

Searching for new mTOR kinase inhibitors: Analysis of binding sites and validation of docking protocols.

The mammalian target of rapamycin (mTOR) is an important biological target for development of novel anticancer drugs and potential antiageing agents. Therefore, many scientific groups search for mTOR kinase inhibitors. Herein, we present structure-based approach which could be helpful in the studies on new bioactive compounds. Method validation was preceded by structural analysis of ATP catalytic cleft and FRB domain. In silico studies allowed us to point crucial amino acid residues for ligand binding and develop optimal docking protocols. The presented methodology could be applied for design and development of potential mTOR kinase inhibitors.

Molecular Mechanisms Underlying the Effect of Lipid Oxidation Products on Digestibility and Conformation of Myoglobin under Thermal Treatment.

Lipid oxidation can produce lipid oxidation products (LOPs), which further react with proteins and affect their structure and digestibility, although the underlying mechanism remains unclear. Herein, we investigated the conformation and digestibility of proteins induced by LOPs after thermal treatment. Digestibility of myoglobin (Mb) affected by trans,trans,-2,4-decadienal (2,4-Dec) was significantly reduced under high temperature (100-180 °C). The peptides digested from Mb modified with 2,4-Dec during thermal processing revealed that the quantity of peptides decreased with increasing 2,4-Dec concentrations. Proteomic analysis showed that 2,4-Dec covalently binds to Mb, and the extent of modification was in the following order: lysine > histidine > arginine. Moreover, the secondary structure, intrinsic fluorescence, and surface hydrophobicity results suggested that 2,4-Dec induced changes in Mb, leading to a tighter spatial structure and aggregation, and exposure of fewer recognition sites of the enzyme and thermal treatment assisted these changes in the structure. Meanwhile, molecular dynamics simulations elucidated the molecular mechanisms underlying the effect of 2,4-Dec and temperature on the digestion and structure of Mb.

CC+ : A searchable database of validated coiled coils in PDB structures and AlphaFold2 models.

α-Helical coiled coils are common tertiary and quaternary elements of protein structure. In coiled coils, two or more α helices wrap around each other to form bundles. This apparently simple structural motif can generate many architectures and topologies. Coiled coil-forming sequences can be predicted from heptad repeats of hydrophobic and polar residues, hpphppp, although this is not always reliable. Alternatively, coiled-coil structures can be identified using the program SOCKET, which finds knobs-into-holes (KIH) packing between side chains of neighboring helices. SOCKET also classifies coiled-coil architecture and topology, thus allowing sequence-to-structure relationships to be garnered. In 2009, we used SOCKET to create a relational database of coiled-coil structures, CC+ , from the RCSB Protein Data Bank (PDB). Here, we report an update of CC+ following an update of SOCKET (to Socket2) and the recent explosion of structural data and the success of AlphaFold2 in predicting protein structures from genome sequences. With the most-stringent SOCKET parameters, CC+ contains ≈12,000 coiled-coil assemblies from experimentally determined structures, and ≈120,000 potential coiled-coil structures within single-chain models predicted by AlphaFold2 across 48 proteomes. CC+ allows these and other less-stringently defined coiled coils to be searched at various levels of structure, sequence, and side-chain interactions. The identified coiled coils can be viewed directly from CC+ using the Socket2 application, and their associated data can be downloaded for further analyses. CC+ is available freely at http://coiledcoils.chm.bris.ac.uk/CCPlus/Home.html. It will be updated automatically. We envisage that CC+ could be used to understand coiled-coil assemblies and their sequence-to-structure relationships, and to aid protein design and engineering.

The Role of Hexon Amino Acid 188 Varies in Fowl Adenovirus Serotype 4 Strains with Different Virulence.

Hepatitis-hydropericardium syndrome (HHS) induced by fowl adenovirus serotype 4 (FAdV-4) has caused huge economic losses to poultry industries. The key genes responsible for different virulence of FAdV-4 strains are not fully elucidated. Previous studies indicated that hexon of pathogenic FAdV-4 has a conserved arginine (R) at position 188, and a conserved isoleucine (I) is present at this position in reported nonpathogenic FAdV-4. Recently, it was reported that R188 of hexon is the determinant site for pathogenicity of the emerging Chinese FAdV-4 strain. However, the role of hexon amino acid 188 (aa188) has not been examined in the nonpathogenic FAdV-4 strain. In this study, three recombinant FAdV-4 viruses, H/H/R188I, O/O/I188R, and H/O/I188R, were constructed by mutating hexon aa188 of FAdV-4 pathogenic strain CH/HNJZ/2015 (H) and nonpathogenic strain ON1 (O), and pathogenicity was assessed in specific-pathogen-free (SPF) chickens. Consistent with previous findings, H/O/I188R exhibited pathogenicity similar to that of CH/HNJZ/2015, yet H/H/R188I induced no mortality. Unexpectedly, all chickens infected with O/O/I188R survived. Postmortem examination of O/O/I188R-infected chickens showed typical lesions of inclusion body hepatitis rather than HHS. Expression of proinflammatory cytokines in CH/HNJZ/2015- and H/O/I188R-infected chickens was significantly higher than that in H/H/R188I-, ON1-, and O/O/I188R-infected chickens. Analysis of predicted hexon protein structures indicated that aa188 mutation leads to conformational changes in the L1 loop of HNJZ-hexon but not in ON1-hexon. In summary, the present study demonstrated that the role of hexon aa188 in the virulence of FAdV-4 varies between different strains. Induction of HHS requires factors aside from hexon aa188 in the emerging Chinese FAdV-4 strain. IMPORTANCE HHS induced by FAdV-4 has caused huge economic losses to the poultry industry. The key determinants for the different virulence of FAdV-4 have not been fully elucidated. Here, we investigated the role of hexon aa188 in FAdV-4 strains with different virulence and showed that the role of hexon aa188 varies in FAdV-4 strains with different genetic contents. The hexon R188 may be the key amino acid for causing inclusion body hepatitis by the pathogenic FAdV-4 strain, and induction of HHS by FAdV-4 may need other viral cofactors. Moreover, the hexon R188I mutation greatly affected the expression of proinflammatory cytokines induced by the pathogenic strain CH/HNJZ/2015, but no significant difference was observed between the nonpathogenic strain ON1 and ON1 with hexon I188R mutation. We found that hexon aa188 mutation induced conformational changes to hexon protein in CH/HNJZ/2015 but not in ON1, which might be the underlying reason for the changing virulence.