Protein Interface Research Articles

RETRACTED ARTICLE: New insights into the microscopic interactions associated with the physical mechanism of action of highly diluted biologics

In this investigation, we report the effect on the microscopic dynamics and interactions of the cytokine interferon gamma (IFN-γ) and antibodies to IFN-γ (anti-IFN-γ) and to the interferon gamma receptor 1 (anti-IFNGR1) prepared in exceptionally dilute solutions of initial proteins. Using both THz spectroscopy and molecular dynamics simulations we have uncovered that the high dilution method of sample preparation results in the reorganization of the sample surface residue dynamics at the solvent–protein interface that leads to both structural and kinetic heterogeneous dynamics that ultimately create interactions that enhance the binding probability of the antigen binding site. Our results indicate that the modified interfacial dynamics of anti-IFN-γ and anti-IFGNR1 that we probe experimentally are directly associated with alterations in the complementarity regions of the distinct antibodies that designate both antigen–antibody affinity and recognition.

Interaction between the transmembrane domains of neurotrophin receptors p75 and TrkA mediates their reciprocal activation

The neurotrophin receptors p75 and tyrosine protein kinase receptor A (TrkA) play important roles in the development and survival of the nervous system. Biochemical data suggest that p75 and TrkA reciprocally regulate the activities of each other. For instance, p75 is able to regulate the response of TrkA to lower concentrations of nerve growth factor (NGF), and TrkA promotes shedding of the extracellular domain of p75 by α-secretases in a ligand-dependent manner. The current model suggests that p75 and TrkA are regulated by means of a direct physical interaction; however, the nature of such interaction has been elusive thus far. Here, using NMR in micelles, multiscale molecular dynamics, FRET, and functional studies, we identified and characterized the direct interaction between TrkA and p75 through their respective transmembrane domains (TMDs). Molecular dynamics of p75-TMD mutants suggests that although the interaction between TrkA and p75 TMDs is maintained upon mutation, a specific protein interface is required to facilitate TrkA active homodimerization in the presence of NGF. The same mutations in the TMD protein interface of p75 reduced the activation of TrkA by NGF as well as reducing cell differentiation. In summary, we provide a structural model of the p75–TrkA receptor complex necessary for neuronal development stabilized by TMD interactions.

Engineered pH-Sensitive Protein G/IgG Interaction.

While natural protein-protein interactions have evolved to be induced by complex stimuli, rational design of interactions that can be switched-on-demand still remain challenging in the protein design world. Here, we demonstrate that a computationally redesigned natural interface for improved binding affinity could further be mutated to adopt a pH switchable interaction. The redesigned interface of Protein G/human IgG Fc domain (referred to as PrG/hIgG), when incorporated with histidine and glutamic acid on PrG (PrG-EHHE), showed a switch in binding affinity by 50-fold when the pH was altered from mild acidic to mild basic. The wild-type (WT) interface showed a negligible switch. The overall binding affinity under mild acidic pH for PrG-EHHE outperformed the wild-type PrG (PrG-WT) interaction. The new reagent PrG-EHHE can be revolutionary in IgG purification, since the standard method of using an extreme acidic pH for elution can be circumvented.

Elaiophylin Is a Potent Hsp90/ Cdc37 Protein Interface Inhibitor with K-Ras Nanocluster Selectivity

The natural product elaiophylin is a macrodiolide with a broad range of biological activities. However, no direct target of elaiophylin in eukaryotes has been described so far, which hinders a systematic explanation of its astonishing activity range. We recently showed that the related conglobatin A, a protein–protein interface inhibitor of the interaction between the N-terminus of Hsp90 and its cochaperone Cdc37, blocks cancer stem cell properties by selectively inhibiting K-Ras4B but not H-Ras. Here, we elaborated that elaiophylin likewise disrupts the Hsp90/ Cdc37 interaction, without affecting the ATP-pocket of Hsp90. Similarly to conglobatin A, elaiophylin decreased expression levels of the Hsp90 client HIF1α, a transcription factor with various downstream targets, including galectin-3. Galectin-3 is a nanocluster scaffold of K-Ras, which explains the K-Ras selectivity of Hsp90 inhibitors. In agreement with this K-Ras targeting and the potent effect on other Hsp90 clients, we observed with elaiophylin treatment a submicromolar IC50 for MDA-MB-231 and MIA-PaCa-2 3D spheroid formation. Finally, a strong inhibition of MDA-MB-231 cells grown in the chorioallantoic membrane (CAM) microtumor model was determined. These results suggest that several other macrodiolides may have the Hsp90/ Cdc37 interface as a target site.

Structure and function at the lipid–protein interface of a pentameric ligand-gated ion channel

Although it has long been proposed that membrane proteins may contain tightly bound lipids, their identity, the structure of their binding sites, and their functional and structural relevance have remained elusive. To some extent, this is because tightly bound lipids are often located at the periphery of proteins, where the quality of density maps is usually poorer, and because they may be outcompeted by detergent molecules used during standard purification procedures. As a step toward characterizing natively bound lipids in the superfamily of pentameric ligand-gated ion channels (pLGICs), we applied single-particle cryogenic electron microscopy to fragments of native membrane obtained in the complete absence of detergent-solubilization steps. Because of the heterogeneous lipid composition of membranes in the secretory pathway of eukaryotic cells, we chose to study a bacterial pLGIC (ELIC) expressed in Escherichia coli's inner membrane. We obtained a three-dimensional reconstruction of unliganded ELIC (2.5-Å resolution) that shows clear evidence for two types of tightly bound lipid at the protein-bulk-membrane interface. One of them was consistent with a "regular" diacylated phospholipid, in the cytoplasmic leaflet, whereas the other one was consistent with the tetra-acylated structure of cardiolipin, in the periplasmic leaflet. Upon reconstitution in E. coli polar-lipid bilayers, ELIC retained the functional properties characteristic of members of this superfamily, and thus, the fitted atomic model is expected to represent the (long-debated) unliganded-closed, "resting" conformation of this ion channel. Notably, the addition of cardiolipin to phosphatidylcholine membranes restored the ion-channel activity that is largely lost in phosphatidylcholine-only bilayers.

Moving pictures: Reassessing docking experiments with a dynamic view of protein interfaces.

The modeling of protein assemblies at the atomic level remains a central issue in structural biology, as protein interactions play a key role in numerous cellular processes. This problem is traditionally addressed using docking tools, where the quality of the models is based on their similarity to a single reference experimental structure. However, using a static reference does not take into account the dynamic quality of the protein interface. Here, we used all-atom classical Molecular Dynamics simulations to investigate the stability of the reference interface for three complexes that previously served as targets in the CAPRI competition. For each one of these targets, we also ran MD simulations for ten models that are distributed over the High, Medium and Acceptable accuracy categories. To assess the quality of these models from a dynamic perspective, we set up new criteria which take into account the stability of the reference experimental protein interface. We show that, when the protein interfaces are allowed to evolve along time, the original ranking based on the static CAPRI criteria no longer holds as over 50% of the docking models undergo a category change (which can be either toward a better or a lower accuracy group) when reassessing their quality using dynamic information.

Bacterial Evolutionary Precursors of Eukaryotic Copper-Zinc Superoxide Dismutases.

Internalization of a bacteria by an archaeal cell expedited eukaryotic evolution. An important feature of the species that diversified into the great variety of eukaryotic life visible today was the ability to combat oxidative stress with a copper–zinc superoxide dismutase (CuZnSOD) enzyme activated by a specific, high-affinity copper chaperone. Adoption of a single protein interface that facilitates homodimerization and heterodimerization was essential; however, its evolution has been difficult to rationalize given the structural differences between bacterial and eukaryotic enzymes. In contrast, no consistent strategy for the maturation of periplasmic bacterial CuZnSODs has emerged. Here, 34 CuZnSODs are described that closely resemble the eukaryotic form but originate predominantly from aquatic bacteria. Crystal structures of a Bacteroidetes bacterium CuZnSOD portray both prokaryotic and eukaryotic characteristics and propose a mechanism for self-catalyzed disulfide maturation. Unification of a bacterial but eukaryotic-like CuZnSOD along with a ferredoxin-fold MXCXXC copper-binding domain within a single polypeptide created the advanced copper delivery system for CuZnSODs exemplified by the human copper chaperone for superoxide dismutase-1. The development of this system facilitated evolution of large and compartmentalized cells following endosymbiotic eukaryogenesis.

Pepscan Approach for the Identification of Protein-Protein Interfaces: Lessons from Experiment.

PEPscan is an old approach that has recently gained renewed interest for the identification of interfering peptides (IPs), i.e., peptides able to interfere with protein–protein interactions (PPIs). Its principle is to slice a protein sequence as a series of short overlapping peptides that are synthesized on a peptide array and tested for their ability to bind a partner, with positive spots corresponding to candidate IPs. PEPscan has been applied with a rather large success in various contexts, but the structural determinants underlying this success remain obscure. Here, we analyze the results of 14 PEPscan experiments, and confront the in vitro results with the available structural information. PEPscan identifies candidate IPs in limited numbers that in all cases correspond to solvent-accessible regions of the structures, their location at the protein–protein interface remaining to be further demonstrated. A strong point of PEPscan seems to be its ability to identify specific IPs. IPs identified from the same protein differ depending on the target PPI, and correspond to patches not frequently involved in the interactions seen in the 3D structures available. Overall, PEPscan seems to provide a cheap and rapid manner to identify candidate IPs, that also comes with room for improvement.

Factor Xa inhibitors: critical considerations for clinical development and testing

The selection of factor (F) X and its activated protease FXa for targeted inhibition to prevent and treat thrombotic conditions is based on an understanding of coagulation biochemistry, sequential steps that occur on tissue factor bearing cells and the interface of coagulation proteins, platelets, mononuclear cells and the nuclear constituents of inflammatory cells. The goal for developing direct oral FXa inhibitors was to achieve rapid, selective, predictable, safe and effective anticoagulation across a broad group of patients expected to derive benefit. The history and development in patient care are exemplars of knowledge, translation and collaboration between the public and private sectors.

Energy Dissipation Hypothesis Applied to Enhance the Affinity of Thrombin Binding Aptamer

Nucleic acid aptamers are artificial recognizing molecules that are capable of specific binding to a wide variety of targets. Aptamers are commonly selected from a huge library of oligonucleotides and improved by introducing several mutations or modular constructions. Although aptamers hold great promise as therapeutic and diagnostic tools, no simple approach to improve their affinity has been suggested yet. Our recent analysis of aptamer–protein complexes revealed that aptamer affinity correlates with the size of an amino acid sidechain in the protein interface that was explained by efficient dissipation of the energy released during complex formation. G-quadruplex-based thrombin aptamers are not involved in the described dependence. Moreover, aptamers to the same thrombin site have 100-fold differences in affinity. Here we focused on a detailed analysis of the nucleic acid interface of thrombin–aptamer complexes. High affinity of the aptamers was shown to correlate with the solvent accessibility of the apolar part of recognizing loops. To prove the concept experimentally, these loops were modified to enhance contact with the solvent. Dissociation rates of the aptamer–thrombin complexes were drastically slowed due to these modifications. In full correspondence with the energy dissipation hypothesis, the modifications improved both the stability of the G-quadruplexes and affinity to thrombin. The most evident effect was shown for unstable Na+-coordinated G-quadruplexes. These data are of high interest for a directed improvement of aptamers introducing unnatural modifications into the ‘hot spot’ residues.

Bridging between material properties of proteins and the underlying molecular interactions.

In this work, we develop a novel method that bridges between material properties of proteins, particularly the modulus of elasticity, and the underlying molecular interactions. To this end, we employ both an all-atom normal mode analysis (NMA) model with the CHARMM force field and an elastic solid model for proteins and protein interfaces. And the "bridge" between the two models is a common physical property predictable by both models: the magnitude of thermal vibrations. This connection allows one to calibrate the Young's moduli of proteins and protein interface regions. We find that the Young's moduli of proteins are in the range of a few Gpa to 10 Gpa, while the Young's moduli of the interface regions are several times smaller. The work is significant as it represents the first attempt to systematically compute the elastic moduli of proteins from molecular interactions.

The virus-host interface: Molecular interactions of Alphacoronavirus-1 variants from wild and domestic hosts with mammalian aminopeptidase N.

The Alphacoronavirus‐1 species include viruses that infect numerous mammalian species. To better understand the wide host range of these viruses, better knowledge on the molecular determinants of virus–host cell entry mechanisms in wildlife hosts is essential. We investigated Alphacoronavirus‐1 infection in carnivores using long‐term data on Serengeti spotted hyenas (Crocuta crocuta) and molecular analyses guided by the tertiary structure of the viral spike (S) attachment protein's interface with the host receptor aminopeptidase N (APN). We sequenced the complete 3′‐end region of the genome of nine variants from wild African carnivores, plus the APN gene of 15 wild carnivore species. Our results revealed two outbreaks of Alphacoronavirus‐1 infection in spotted hyenas associated with genetically distinct canine coronavirus type II (CCoVII) variants. Within the receptor binding domain (RBD) of the S gene the residues that directly bind to the APN receptor were conserved in all variants studied, even those infecting phylogenetically diverse host taxa. We identified a variable region within RBD located next to a region that directly interacts with the APN receptor. Two residues within this variable region were under positive selection in hyena variants, indicating that both sites were associated with adaptation of CCoVII to spotted hyena APN. Analysis of APN sequences revealed that most residues that interact with the S protein are conserved in wild carnivores, whereas some adjacent residues are highly variable. Of the variable residues, four that are critical for virus–host binding were under positive selection and may modulate the efficiency of virus attachment to carnivore APN.

Analysis of Binding Modes of Antigen-Antibody Complexes by Molecular Mechanics Calculation.

Antibodies are one of the most important protein molecules in biopharmaceutics. Due to the recent advance in technology for producing monoclonal antibodies, many structural data are available on the antigen-antibody complexes. To characterize the molecular interaction in antigen-antibody recognition, we computationally analyzed 500 complex structures by molecular mechanics calculations. The presence of Ser and Tyr is markedly large in the complementarity-determining regions (CDRs). Although Ser is abundant in CDRs, its contribution to the binding score is not large. Instead, Tyr, Asp, Glu, and Arg significantly contribute to the molecular interaction from the viewpoint of the binding score. The decomposition of the binding score suggests that the hydrophilic interaction is predominant in all CDRs compared with the hydrophobic one. The contribution of the heavy chain is larger than that of the light chain. In particular, H2 and H3 largely contribute to the binding interaction. Tyr is a main contributing residue both in H2 and H3. The positively charged residue Arg also significantly contributes to the binding score in H3, while the contribution of Lys is small. The appearance of Ser is remarkable in H2, and Asp is abundant in H3. The non-charged polar residues, Thr, Asn, and Gln, appear much in H2, compared to appearing in H3. The negatively charged residues Asp and Glu significantly contribute to the binding score in H3. The contributions of Phe and Trp are not large in spite that the aromatic residues are capable of making the π-π or CH-π interaction. Gly is commonly abundant both in H2 and H3. The average distance of the shortest direct hydrogen bond between the antigen and antibody is longer than that of the hydrogen bonds observed in the complexes between compounds and their target proteins. Therefore, the antigen-antibody interface is not so tight as the compound-target protein interface. The calculation of shape complementarity is consistent with the result of the hydrogen bonds in that the fitness of the antigen-antibody contact is not so high as that of the compound-target protein contact. There exist many water molecules at the antigen-antibody interface. These findings suggest that Tyr, Asp, Glu, and Arg are rich in H3 and work as major contributors for the interaction with the antigen. Ser, Thr, Asn, and Gln are rich in H2 and support the interaction with enhancing molecular fitness. Gly is helpful in increasing flexibility and geometrical diversity. Because the antigen-antibody binding is fundamentally hydrophilic-driven, the non-polar residues are unfavorable for mediating the contact even for the aromatic residues such as Phe and Trp.

Detection of Binding Sites on SARS-CoV-2 Spike Protein Receptor-Binding Domain by Molecular Dynamics Simulations in Mixed Solvents.

The novel SARS-CoV-2 uses ACE2 (Angiotensin-Converting Enzyme 2) receptor as an entry point. Insights on S protein receptor-binding domain (RBD) interaction with ACE2 receptor and drug repurposing has accelerated drug discovery for the novel SARS-CoV-2 infection. Finding small molecule binding sites in S protein and ACE2 interface is crucial in search of effective drugs to prevent viral entry. In this study, we employed molecular dynamics simulations in mixed solvents together with virtual screening to identify small molecules that could be potential inhibitors of S protein –ACE2 interaction. Observation of organic probe molecule localization during the simulations revealed multiple sites at the S protein surface related to small molecule, antibody, and ACE2 binding. In addition, a novel conformation of the S protein was discovered that could be stabilized by small molecules to inhibit attachment to ACE2. The most promising binding site on RBD-ACE2 interface was targeted with virtual screening and top-ranked compounds (DB08248, DB02651, DB03714, and DB14826) are suggested for experimental testing. The protocol described here offers an extremely fast method for characterizing key proteins of a novel pathogen and for the identification of compounds that could inhibit or accelerate spreading of the disease.

Biophysical Compatibility of a Heterotrimeric Tyrosinase-TYRP1-TYRP2 Metalloenzyme Complex.

Tyrosinase (TYR) is a copper-containing monooxygenase central to the function of melanocytes. Alterations in its expression or activity contribute to variations in skin, hair and eye color, and underlie a variety of pathogenic pigmentary phenotypes, including several forms of oculocutaneous albinism (OCA). Many of these phenotypes are linked to individual missense mutations causing single nucleotide variants and polymorphisms (SNVs) in TYR. We previously showed that two TYR homologues, TYRP1 and TYRP2, modulate TYR activity and stabilize the TYR protein. Accordingly, to investigate whether TYR, TYRP1, and TYRP2 are biophysically compatible with various heterocomplexes, we computationally docked a high-quality 3D model of TYR to the crystal structure of TYRP1 and to a high-quality 3D model of TYRP2. Remarkably, the resulting TYR-TYRP1 heterodimer was complementary in structure and energy with the TYR-TYRP2 heterodimer, with TYRP1 and TYRP2 docking to different adjacent surfaces on TYR that apposed a third realistic protein interface between TYRP1-TYRP2. Hence, the 3D models are compatible with a heterotrimeric TYR-TYRP1-TYRP2 complex. In addition, this heterotrimeric TYR-TYRP1-TYRP2 positioned the C-terminus of each folded enzymatic domain in an ideal position to allow their C-terminal transmembrane helices to form a putative membrane embedded three-helix bundle. Finally, pathogenic TYR mutations causing OCA1A, which also destabilize TYR biochemically, cluster on an unoccupied protein interface at the periphery of the heterotrimeric complex, suggesting that this may be a docking site for OCA2, an anion channel. Pathogenic OCA2 mutations result in similar phenotypes to those produced by OCA1A TYR mutations. While this complex may be difficult to detect in vitro, due to the complex environment of the vertebrate cellular membranous system, our results support the existence of a heterotrimeric complex in melanogenesis.

Atomic-level evolutionary information improves protein-protein interface scoring.

The crucial role of protein interactions and the difficulty in characterizing them experimentally strongly motivates the development of computational approaches for structural prediction. Even when protein-protein docking samples correct models, current scoring functions struggle to discriminate them from incorrect decoys. The previous incorporation of conservation and coevolution information has shown promise for improving protein-protein scoring. Here, we present a novel strategy to integrate atomic-level evolutionary information into different types of scoring functions to improve their docking discrimination. We applied this general strategy to our residue-level statistical potential from InterEvScore and to two atomic-level scores, SOAP-PP and Rosetta interface score (ISC). Including evolutionary information from as few as 10 homologous sequences improves the top 10 success rates of individual atomic-level scores SOAP-PP and Rosetta ISC by 6 and 13.5 percentage points, respectively, on a large benchmark of 752 docking cases. The best individual homology-enriched score reaches a top 10 success rate of 34.4%. A consensus approach based on the complementarity between different homology-enriched scores further increases the top 10 success rate to 40%. All data used for benchmarking and scoring results, as well as a Singularity container of the pipeline, are available at http://biodev.cea.fr/interevol/interevdata/. Supplementary data are available at Bioinformatics online.

An ultra-high affinity protein-protein interface displaying sequence-robustness.

Protein-protein interactions are crucial in biology and play roles in for example, the immune system, signaling pathways, and enzyme regulation. Ultra-high affinity interactions (Kd <0.1nM) occur in these systems, however, structures and energetics behind stability of ultra-high affinity protein-protein complexes are not well understood. Regulation of the starch debranching barley limit dextrinase (LD) and its endogenous cereal type inhibitor (LDI) exemplifies an ultra-high affinity complex (Kd of 42 pM). In this study the LD-LDI complex is investigated to unveil how robust the ultra-high affinity is to LDI sequence variation at the protein-protein interface and whether alternative sequences can retain the ultra-high binding affinity. The interface of LD-LDI was engineered using computational protein redesign aiming at identifying LDI variants predicted to retain ultra-high binding affinity. These variants present a very diverse set of mutations going beyond conservative and alanine substitutions typically used to probe interfaces. Surface plasmon resonance analysis of the LDI variants revealed that high affinity of LD-LDI requires interactions of several residues at the rim of the protein interface, unlike the classical hotspot arrangement where key residues are found at the center of the interface. Notably, substitution of interface residues in LDI, including amino acids with functional groups different from the wild-type, could occur without loss of affinity. This demonstrates that ultra-high binding affinity can be conferred without hotspot residues, thus making complexes more robust to mutational drift in evolution. The present mutational analysis also demonstrates how energetic coupling can emerge between residues at large distances at the interface.

Mediator subunit Med15 dictates the conserved \u201cfuzzy\u201d binding mechanism of yeast transcription activators Gal4 and Gcn4

The acidic activation domain (AD) of yeast transcription factor Gal4 plays a dual role in transcription repression and activation through binding to Gal80 repressor and Mediator subunit Med15. The activation function of Gal4 arises from two hydrophobic regions within the 40-residue AD. We show by NMR that each AD region binds the Mediator subunit Med15 using a “fuzzy” protein interface. Remarkably, comparison of chemical shift perturbations shows that Gal4 and Gcn4, two intrinsically disordered ADs of different sequence, interact nearly identically with Med15. The finding that two ADs of different sequence use an identical fuzzy binding mechanism shows a common sequence-independent mechanism for AD-Mediator binding, similar to interactions within a hydrophobic cloud. In contrast, the same region of Gal4 AD interacts strongly with Gal80 via a distinct structured complex, implying that the structured binding partner of an intrinsically disordered protein dictates the type of protein–protein interaction.

Molecular Docking and Dynamics Simulation of a Screening Library from Life Chemicals Database for Potential Protein-Protein Interactions (PPIs) Inhibitors against SARS-CoV-2 Spike Protein

The ongoing pandemic of coronavirus 2 represents a major challenge for global public health authorities. Coronavirus disease 2019 can be fatal especially in elderly people and those with comorbidities. Currently, several vaccines against coronavirus 2 are under application in multiple countries with emergency use authorization. In the same time, many vaccine candidates are under development and assessment. It is worth noting that the design of some of these vaccines depends on the expression of receptor binding domain for viral spike protein to induce host immunity. As such, blocking the spike protein interface with antibodies, peptides or small molecular compounds can impede the ability of coronavirus 2 to invade host cells by intervention with interactions between viral spike protein and cellular angiotensin converting enzyme 2. In this virtual screening study, we have used predictive webservers, molecular docking and dynamics simulation to evaluate the ability of 3000 compounds to interact with interface residues of spike protein receptor binding domain. This library of chemicals was focused by Life Chemicals as potential protein-protein interactions inhibitor. Here, we report that hit compound 7, with IUPAC name of 3‐cyclohexyl‐N‐(4‐{[(1R,9R) ‐6‐oxo‐7,11‐ diazatricyclo [7.3.1.02,7] trideca‐2,4‐dien‐11‐yl] sulfonyl} phenyl) propenamide, may have the capacity to interact with interface of receptor binding domain for viral spike protein and thereby reduce cellular entry of the virus. However, in vitro and in vivo assessments may be required to validate these virtual findings.

Identification of a Novel Cis-Acting Regulator of HIV-1 Genome Packaging.

Human immunodeficiency virus type 1 (HIV-1) uptakes homo-dimerized viral RNA genome into its own particle. A cis-acting viral RNA segment responsible for this event, termed packaging signal (psi), is located at the 5′-end of the viral genome. Although the psi segment exhibits nucleotide variation in nature, its effects on the psi function largely remain unknown. Here we show that a psi sequence from an HIV-1 regional variant, subtype D, has a lower packaging ability compared with that from another regional variant, HIV-1 subtype B, despite maintaining similar genome dimerization activities. A series of molecular genetic investigations narrowed down the responsible element of the selective attenuation to the two sequential nucleotides at positions 226 and 227 in the psi segment. Molecular dynamics simulations predicted that the dinucleotide substitution alters structural dynamics, fold, and hydrogen-bond networks primarily of the psi-SL2 element that contains the binding interface of viral nucleocapsid protein for the genome packaging. In contrast, such structural changes were minimal within the SL1 element involved in genome dimerization. These results suggest that the psi 226/227 dinucleotide pair functions as a cis-acting regulator to control the psi structure to selectively tune the efficiency of packaging, but not dimerization of highly variable HIV-1 genomes.