Self-assembled peptide-based biomaterials provide versatile platforms for biomedical uses, featuring customizable physicochemical properties, biocompatibility, and dynamic capabilities. This self-assembly process is primarily dictated by primary sequence features, such as hydrophobicity, length, and charge, leading to the formation of fibrils and hydrogels. Amphipathic peptides, with alternating polar and hydrophobic residues, are especially effective in forming supramolecular nanofibers stabilized by π-π interactions and hydrogen bonds. Chemical modifications on aromatic side chains are promising for controlling assembly morphology, stability, and biological activity. However, the influence of these substituents on peptide packing and immunogenicity remains relatively unexplored. Herein, we examine the effect of substituents on benzyl groups attached to short amphipathic peptides. By introducing different electron-donating and withdrawing groups at the para-position of benzyl rings and modifying the chain length connecting the backbone to the aromatic moiety, we observe notable effects on fibril formation, molecular packing, and immunogenicity both in vitro and in vivo. Our results show that subtle chemical modifications are practical tools for designing tailored peptide nanomaterials with promising potential in vaccine delivery, tissue engineering, and regenerative medicine.

Peptide coassembly and self-assembly are complex processes that enable the formation of intricate nanoscale structures, facilitating the development of novel functional biomaterials. In this study, we explore stereocomplexation-driven enantiomeric coassembly, focusing on the distinctive structures of 'block heterochiral' peptides. Using the model amphipathic peptide KFE8 (FKFEFKFE), which consists of two FKFE blocks, we created homochiral peptides made of repeat units with the same chirality (LL and DD) or heterochiral variants with opposite chirality units (LD and DL). We examined the pairwise assembly behaviors of these identical peptides, which differ only in segmented stereoisomerization. We analyzed the nano-, micro-, and macroscale properties of LL/LD, LL/DL, DD/LD, and DD/DL, each representing two equimolar stereoisomers mixed before solubilization ('premixed') to enable potential coassembly or self-sorting within a single, well-mixed monomer phase. We also used 'postmixed systems', which were assembled separately and combined only before data collection for each technique (e.g., LL+LD) as controls. Our data show that homochiral or heterochiral peptide pairs with matching N-terminal chirality coassemble, while a mismatch in N-terminal chirality promotes self-sorting behavior.

Glycosaminoglycans (GAGs) play diverse and fundamental roles in physiology by regulating the function of large classes of proteins. Despite their importance, knowledge of how GAGs are organized in protein-bound complexes remains limited. This can be attributed to the linear structure, conformational flexibility, and high negative charge of GAGs, all of which disfavor structure determination by crystallography or NMR spectroscopy. A hybrid approach based on GAG-binding-induced changes in NMR protein chemical shifts, computational docking, and molecular dynamics simulations has proven to be valuable in providing structural models. However, these approaches can identify multiple plausible GAG geometries, making it difficult to determine whether the observed geometries reflect intrinsic plasticity or limitations of the NMR data and docking methods. In the case of chemokine CXCL8, two GAG-binding modes have been proposed, one within a monomer and the other across the dimer interface. Here, we determined low-resolution solution structures of heparin and chondroitin sulfate octasaccharides bound to the CXCL8 dimer using small-angle X-ray scattering (SAXS). SAXS analyses show that both heparin and chondroitin sulfate bind to a surface within a monomer and are incompatible with binding across the dimer. NMR paramagnetic relaxation enhancement measurements for heparin-bound CXCL8 dimer and monomer complexes show that heparin engages a similar surface within the monomer in both complexes, consistent with the SAXS models. Together, these studies establish how GAGs are organized in the CXCL8-bound complex and highlight the value of complementary low-resolution structural methods for characterizing GAG-protein complexes.

Peptide-based biomaterials assembled through monomer-by-monomer self-assembly provide versatile platforms for biomedical applications due to their adjustable physicochemical properties, biocompatibility, and dynamic nature. The self-assembly process largely depends on primary sequence features, such as hydrophobicity, length, and charge, which influence the formation of various nanostructures, including fibrils and hydrogels. Amphipathic peptides, characterized by alternating polar and hydrophobic residues, are especially effective in forming supramolecular nanofibers stabilized by π-π interactions and hydrogen bonds. Chemical modifications, particularly on aromatic side chains, have proven to be a promising approach for controlling assembly morphology, stability, and biological activity. In organic chemistry, the use of chemical substituents, such as halogens, alkyl groups, or electron-donating and electron-withdrawing groups, has been widely employed to alter reactivity, stability, and molecular interactions for diverse applications, including catalysts, pharmaceuticals, and materials science. However, the influence of these substituents on peptide packing and in vivo immunogenicity remains relatively unexplored. In this study, we systematically examine how changes in the position and nature of substituents on benzyl groups attached to short amphipathic peptides affect self-assembly, fibril morphology, and immune responses. By introducing different electron-donating and withdrawing groups at the para-position of benzyl rings and modifying the chain length connecting the backbone to the aromatic moiety, we observe notable effects on fibril formation, molecular packing, and immunogenicity both in vitro and in vivo . Our results show that subtle chemical modifications are effective tools for designing tailored peptide nanomaterials with promising potential in vaccine delivery, tissue engineering, and regenerative medicine.

The large dsDNA viruses replicate their DNA as concatemers consisting of multiple covalently linked genomes. Genome packaging is catalyzed by a terminase enzyme that excises individual genomes from concatemers and packages them into preassembled procapsids. These disparate tasks are catalyzed by terminase alternating between two distinct states-a stable nuclease that excises individual genomes and a dynamic motor that translocates DNA into the procapsid. It was proposed that bacteriophage λ terminase assembles as an anti-parallel dimer-of-dimers nuclease complex at the packaging initiation site. In contrast, all characterized packaging motors are composed of five terminase subunits bound to the procapsid in a parallel orientation. Here, we describe biophysical and structural characterization of the λ holoenzyme complex assembled in solution. Analytical ultracentrifugation, small angle X-ray scattering, and native mass spectrometry indicate that 5 subunits assemble a cone-shaped terminase complex. Classification of cryoEM images reveals starfish-like rings with skewed pentameric symmetry and one special subunit. We propose a model wherein nuclease domains of two subunits alternate between a dimeric head-to-head arrangement for genome maturation and a fully parallel arrangement during genome packaging. Given that genome packaging is strongly conserved in both prokaryotic and eukaryotic viruses, the results have broad biological implications.

Protein SUMOylation is a prevalent stress-response posttranslational modification crucial for maintaining cellular homeostasis. Herein, we report that protein SUMOylation modulates cellular signaling mediated by cAMP, an ancient and universal stress-response second messenger. We identify K561 as a primary SUMOylation site in exchange protein directly activated by cAMP (EPAC1) via site-specific mapping of SUMOylation using mass spectrometry. Sequence and site-directed mutagenesis analyses reveal that a functional SUMO-interacting motif in EPAC1 is required for the binding of SUMO-conjugating enzyme UBC9, formation of EPAC1 nuclear condensate, and EPAC1 cellular SUMOylation. Heat shock-induced SUMO modification of EPAC1 promotes Rap1/2 activation in a cAMP-independent manner. Structural modeling and molecular dynamics simulation studies demonstrate that SUMO substituent on K561 of EPAC1 promotes Rap1 interaction by increasing the buried surface area between the SUMOylated receptor and its effector. Our studies identify a functional SUMOylation site in EPAC1 and unveil a novel mechanism in which SUMOylation of EPAC1 leads to its autonomous activation. The findings of SUMOylation-mediated activation of EPAC1 not only provide new insights into our understanding of cellular regulation of EPAC1 but also will open up a new field of experimentation concerning the cross-talk between cAMP/EPAC1 signaling and protein SUMOylation, two major cellular stress response pathways, during cellular homeostasis.

Abstract The genomes of positive-strand RNA viruses serve as a template for both protein translation and genome replication. In enteroviruses, a cloverleaf RNA structure at the 5′ end of the genome functions as a switch to transition from viral translation to replication by interacting with host poly(C)-binding protein 2 (PCBP2) and the viral 3CDpro protein. We determined the structures of cloverleaf RNA from coxsackievirus and poliovirus. Cloverleaf RNA folds into an H-type four-way junction and is stabilized by a unique adenosine-cytidine-uridine (A•C-U) base triple involving the conserved pyrimidine mismatch region. The two PCBP2 binding sites are spatially proximal and are located on the opposite end from the 3CDpro binding site on cloverleaf. We determined that the A•C-U base triple restricts the flexibility of the cloverleaf stem–loops resulting in partial occlusion of the PCBP2 binding site, and elimination of the A•C-U base triple increases the binding affinity of PCBP2 to the cloverleaf RNA. Based on the cloverleaf structures and biophysical assays, we propose a new mechanistic model by which enteroviruses use the cloverleaf structure as a molecular switch to transition from viral protein translation to genome replication.

Like many dsDNA viruses, bacteriophage λ replicates its genome as a concatemer consisting of multiple copies of covalently linked dsDNA genomes. To encapsidate a single genome within a nascent procapsid, λ must: 1) find its own dsDNA amongst the multitude of host nucleic acids; 2) identify the genomic start site; 3) cut the DNA; 4) bring the excised DNA to a procapsid; 5) translocate DNA into the capsid; 6) cut DNA again at a packaging termination site, 7) disengage from the newly filled capsid; and 8) bring the remainder of the genomic concatemer to fill another empty procapsid. These disparate genome processing tasks are carried out by a single virus-encoded enzyme complex called terminase. While it has been shown that λ terminase initially forms a tetrameric complex to cut DNA, it is not clear whether the same configuration translocates DNA. Here, we describe biophysical and initial structural characterization of a λ terminase translocation complex. Analytical ultracentrifugation (AUC) and small angle X-ray scattering (SAXS) indicate that between 4 and 5 protomeric subunits assemble a cone-shaped terminase complex with a maximum dimension of ∼230 and radius of gyration of ∼72 Å. Two-dimensional classification of cryoEM images of λ terminase are consistent with these dimensions and show that particles assume a preferred orientation in ice. The orientations appear to be end-on, as terminase rings resemble a starfish with approximate pentameric symmetry. While ∼5-fold symmetry is apparent, one of the five “arms” appears partially displaced with weaker more diffuse density in some classes, suggesting flexibility and/or partial occupancy. Charge detection mass spectrometry ( CDMS ) is consistent with a pentameric complex, with evidence that one motor subunit is weakly bound. Kinetic analysis indicates that the complex hydrolyzes ATP at a rate comparable to the rates of other phage packaging motors. Together with previously published data, these results suggest that λ terminase assembles conformationally and stoichiometrically distinct complexes to carry out different genome processing tasks. We propose a “symmetry resolution” pathway to explain how terminase transitions between these structurally and functionally distinct states.

Protein SUMOylation plays an essential role in maintaining cellular homeostasis when cells are under stress. However, precisely how SUMOylation is regulated, and a molecular mechanism linking cellular stress to SUMOylation, remains elusive. Here, we report that cAMP, a major stress-response second messenger, acts through Epac1 as a regulator of cellular SUMOylation. The Epac1-associated proteome is highly enriched with components of the SUMOylation pathway. Activation of Epac1 by intracellular cAMP triggers phase separation and the formation of nuclear condensates containing Epac1 and general components of the SUMOylation machinery to promote cellular SUMOylation. Furthermore, genetic knockout of Epac1 obliterates oxidized low-density lipoprotein-induced cellular SUMOylation in macrophages, leading to suppression of foam cell formation. These results provide a direct nexus connecting two major cellular stress responses to define a molecular mechanism in which cAMP regulates the dynamics of cellular condensates to modulate protein SUMOylation.

Molecular segregation and biopolymer manipulation require the action of molecular motors to do work by applying directional forces to macromolecules. The additional strand conserved E (ASCE) ring motors are an ancient family of molecular motors responsible for diverse biological polymer manipulation tasks. Viruses use ASCE segregation motors to package their genomes into their protein capsids and provide accessible experimental systems due to their relative simplicity. We show by cryo-EM-focused image reconstruction that ASCE ATPases in viral double-stranded DNA (dsDNA) packaging motors adopt helical symmetry complementary to their dsDNA substrates. Together with previous data, our results suggest that these motors cycle between helical and planar configurations, providing a possible mechanism for directional translocation of DNA. Similar changes in quaternary structure have been observed for proteasome and helicase motors, suggesting an ancient and common mechanism of force generation that has been adapted for specific tasks over the course of evolution.

Double-stranded DNA viruses package their genomes into pre-assembled protein procapsids. This process is driven by macromolecular motors that transiently assemble at a unique vertex of the procapsid and utilize homomeric ring ATPases to couple genome encapsidation to ATP hydrolysis. Here, we describe the biochemical and biophysical characterization of the packaging ATPase from Lactococcus lactis phage asccφ28. Size-exclusion chromatography (SEC), analytical ultracentrifugation (AUC), small angle X-ray scattering (SAXS), and negative stain transmission electron microscopy (TEM) indicate that the ~45 kDa protein formed a 443 kDa cylindrical assembly with a maximum dimension of ~155 Å and radius of gyration of ~54 Å. Together with the dimensions of the crystallographic asymmetric unit from preliminary X-ray diffraction experiments, these results indicate that gp11 forms a decameric D5-symmetric complex consisting of two pentameric rings related by 2-fold symmetry. Additional kinetic analysis shows that recombinantly expressed gp11 has ATPase activity comparable to that of functional ATPase rings assembled on procapsids in other genome packaging systems. Hence, gp11 forms rings in solution that likely reflect the fully assembled ATPases in active virus-bound motor complexes. Whereas ATPase functionality in other double-stranded DNA (dsDNA) phage packaging systems requires assembly on viral capsids, the ability to form functional rings in solution imparts gp11 with significant advantages for high-resolution structural studies and rigorous biophysical/biochemical analysis.

During the life cycle of enteric bacterium Escherichia coli, it encounters a wide spectrum of pH changes. The asymmetric dimer of the cAMP receptor protein, CRP, plays a key role in regulating the expressions of genes and the survival of E. coli. To elucidate the pH effects on the mechanism of signal transmission, we present a combination of results derived from ITC, crystallography, and computation. CRP responds to a pH change by inducing a differential effect on the affinity for the binding events to the two cAMP molecules, ensuing in a reversible conversion between positive and negative cooperativity at high and low pH, respectively. The structures of four crystals at pH ranging from 7.8 to 6.5 show that CRP responds by inducing a differential effect on the structures of the two subunits, particularly in the DNA binding domain. Employing the COREX/BEST algorithm, computational analysis shows the change in the stability of residues at each pH. The change in residue stability alters the connectivity between residues including those in cAMP and DNA binding sites. Consequently, the differential impact on the topology of the connectivity surface among residues in adjacent subunits is the main reason for differential change in affinity; that is, the pH-induced differential change in residue stability is the biothermodynamic basis for the change in allosteric behavior. Furthermore, the structural asymmetry of this homodimer amplifies the differential impact of any perturbations. Hence, these results demonstrate that the combination of these approaches can provide insights into the underlying mechanism of an apparent complex allostery signal and transmission in CRP.

Double-stranded DNA viruses package their genomes into pre-assembled capsids using virally-encoded ASCE ATPase ring motors. We present the first atomic-resolution crystal structure of a multimeric ring form of a viral dsDNA packaging motor, the ATPase of the asccφ28 phage, and characterize its atomic-level dynamics via long timescale molecular dynamics simulations. Based on these results, and previous single-molecule data and cryo-EM reconstruction of the homologous φ29 motor, we propose an overall packaging model that is driven by helical-to-planar transitions of the ring motor. These transitions are coordinated by inter-subunit interactions that regulate catalytic and force-generating events. Stepwise ATP binding to individual subunits increase their affinity for the helical DNA phosphate backbone, resulting in distortion away from the planar ring towards a helical configuration, inducing mechanical strain. Subsequent sequential hydrolysis events alleviate the accumulated mechanical strain, allowing a stepwise return of the motor to the planar conformation, translocating DNA in the process. This type of helical-to-planar mechanism could serve as a general framework for ring ATPases.

Dengue virus (DENV) was designated as a top 10 public health threat by the World Health Organization in 2019. No clinically approved anti-DENV drug is currently available. Here we report the high-resolution cocrystal structure (1.5 Å) of the DENV-2 capsid protein in complex with an inhibitor that potently suppresses DENV-2 but not other DENV serotypes. The inhibitor induces a "kissing" interaction between two capsid dimers. The inhibitor-bound capsid tetramers are assembled inside virions, resulting in defective uncoating of nucleocapsid when infecting new cells. Resistant DENV-2 emerges through one mutation that abolishes hydrogen bonds in the capsid structure, leading to a loss of compound binding. Structure-based analysis has defined the amino acids responsible for the inhibitor's inefficacy against other DENV serotypes. The results have uncovered an antiviral mechanism through inhibitor-induced tetramerization of the viral capsid and provided essential structural and functional knowledge for rational design of panserotype DENV capsid inhibitors.

Many viruses use pentameric assemblies of packaging ATPases to translocate their genome into preformed viral procapsids during assembly. Although several monomeric packaging ATPase structures have been solved, these structures do not reveal how subunits interact with their substrate, double-stranded DNA (dsDNA), or coordinate with each other in order to achieve processive translocation. Here we present the first-ever solved crystal structure of a packaging ATPase pentameric assembly. The structure reveals that inter-subunit contacts are largely mediated by an extended lid subdomain and each subunit's ATPase active site lies at this interface, allowing for residues to be donated in-trans to sense nucleotide occupancy and catalyze ATP hydrolysis. Additionally, the pore is asymmetric and lined by positively charged residues donated by the ATPase domain. We interrogate the dynamics of the assembly in apo, ATP-bound, and ADP-bound states with bound dsDNA using microsecond-scale all-atom molecular dynamics simulations. Our simulations predict that nucleotide-binding actuates rotation of the lid subdomain, consistent with prior predictions. We highlight the distinction between a trans-acting arginine which we predict is used for nucleotide sensing and exchange, and a trans-acting lysine which we predict is used for catalysis. We predict that positively charged residues in the pore are donated to grip dsDNA in direct response to nucleotide binding, and that the observed lid subdomain rotation may be able to modulate the asymmetry of the pore to accommodate the helicity of dsDNA.

UNC-45B is a multidomain molecular chaperone that is essential for the proper folding and assembly of myosin into muscle thick filaments in vivo. It has previously been demonstrated that the UCS domain is responsible for the chaperone-like properties of the UNC-45B. To better understand the chaperoning function of the UCS domain of the UNC-45B chaperone, we engineered mutations designed to 1) disrupt chaperone-client interactions by removing and altering the structure of a putative client-interacting loop and 2) disrupt chaperone-client interactions by changing highly conserved residues in a putative client-binding groove. We tested the effect of these mutations by using a, to our knowledge, novel combination of complementary biophysical assays (circular dichroism, chaperone activity, and small-angle x-ray scattering) and in vivo tools (Caenorhabditis elegans sarcomere structure). Removing the putative client-binding loop altered the secondary structure of the UCS domain (by decreasing the α-helix content), leading to a significant change in its solution conformation and a reduced chaperoning function. Additionally, we found that mutating several conserved residues in the putative client-binding groove did not alter the UCS domain secondary structure or structural stability but reduced its chaperoning activity. In vivo, these groove mutations were found to significantly alter the structure and organization of C. elegans sarcomeres. Furthermore, we tested the effect of R805W, a mutation distant from the putative client-binding region, which in humans, has been known to cause congenital and infantile cataracts. Our in vivo data show that, to our surprise, the R805W mutation appeared to have the most drastic detrimental effect on the structure and organization of the worm sarcomeres, indicating a crucial role of R805 in UCS-client interactions. Hence, our experimental approach combining biophysical and biological tools facilitates the study of myosin-chaperone interactions in mechanistic detail.

Patterned substitution of d-amino acids into the primary sequences of self-assembling peptides influences molecular-level packing and supramolecular morphology. We report that block heterochiral analogs of the model amphipathic peptide KFE8 (Ac-FKFEFKFE-NH2), composed of two FKFE repeat motifs with opposite chirality, assemble into helical tapes with dimensions greatly exceeding those of their fibrillar homochiral counterparts. At sufficient concentrations, these tapes form hydrogels with reduced storage moduli but retain the shear-thinning behavior and consistent mechanical recovery of the homochiral analogs. Varying the identity of charged residues (FRFEFRFE and FRFDFRFD) produced similarly sized nonhelical tapes, while a peptide with nonenantiomeric l- and d-blocks (FKFEFRFD) formed helical tapes closely resembling those of the heterochiral KFE8 analogs. A proposed energy-minimized model suggests that a kink at the interface between l- and d-blocks leads to the assembly of flat monolayers with nonidentical surfaces that display alternating stacks of hydrophobic and charged groups.

Hemoglobin functions as an oxygen transport protein, with each subunit containing a heme cofactor. We have developed a global disassembly model for human hemoglobin, linking hemin (ferric heme) disassociation and apo(heme-free)-protein unfolding pathways. The model was based on the evaluation of circular dichroism and visible absorbance measurements of guanidine hydrochloride-induced disassembly of holo (heme-bound)-hemoglobin and previous measurements of apohemoglobin unfolding. The populations of holo-intermediates and equilibrium disassembly parameters were determined quantitatively for adult and fetal hemoglobins. The key stages for disassembly into unfolded monomers are characterized by hemichrome intermediates with molten globule characteristics. Hemichromes, which occur when both hemin iron axial sites coordinate amino acids, are not energetically favored in native human hemoglobins. However, these hexacoordinate iron complexes are important for preventing hemin disassociation from partially unfolded species during early disassembly and late stage assembly events. Both our model evaluation and independent small angle X-ray scattering measurements demonstrate that heme disassociation during early disassembly leads to loss of tetrameric structural integrity. Dimeric and monomeric hemichrome intermediates occur along the disassembly pathway inside red cells where the hemoglobin concentration is very high. This prediction explains why in the red cells of patients with unstable hemoglobinopathies, misassembled hemoglobins often get trapped as hemichromes that accumulate into insoluble Heinz bodies. These Heinz bodies become deposited on the cell membranes and can lead to hemolysis. Alternatively, when acellular hemoglobin is diluted into blood plasma after red cell lysis, the disassembly pathway is dominated by early hemin disassociation events, which leads to the generation of higher fractions of apo-subunits and free hemin known to damage to the integrity of blood vessel walls. Thus, our model illuminates the pathophysiology of hemoglobinopathies and other disease states associated with unstable globins and red cell lysis, and provides insights into the factors governing hemoglobin assembly during erythropoiesis. Significance Our deconvolution and global analysis of spectral data led to both the characterization of “hidden” hemichrome intermediates and the development of a quantitative model for human hemoglobin disassembly/assembly. The importance of this mechanism is several-fold. First, the hemoglobin system serves as a general biological model for understanding the role of oligomerization and cofactor binding in facilitating protein folding and assembly. Second, the fitted parameters provide: (a) estimates of hemin affinity for apoprotein states; (b) quantitative interpretations of the pathophysiology of hemoglobinopathies and other diseases associated with unstable globins and red cell lysis; (c) insights into the factors governing hemoglobin assembly during erythropoiesis; and (d) a framework for designing targeted hemoglobinopathy therapeutics.