The conformational dynamics of Hepatitis C Virus E2 glycoprotein with the increasing number of N-glycosylation unraveled by molecular dynamics simulations.

The Hepatitis C Virus (HCV), responsible for causing hepatitis and a significant contributor to liver disorders, presents a challenge for treatment due to its high genetic variability. Despite efforts, there is still no effective medication available for this virus. One of the promising targets for drug development involves targeting glycoprotein E2. However, our understanding of the dynamic behavior of E2 and its associated glycans remains limited. In this study, we investigated the dynamic characteristics of E2 with varying degrees of glycosylation using all-atom molecular dynamics simulations. We also explored glycan's interactions with the protein and among themselves. An overall increase in correlation between the vital protein regions was observed with an increase in glycan number. The protein dynamics is followed by the analysis of glycan dynamics, where the flexibility of the individual glycans was analyzed in their free and bound state, which revealed a decrease in their fluctuation in some cases. Furthermore, we generated the free energy landscape of individual N-glycan linkages in both free and bound states and observed both increases and decreases in flexibility, which can be attributed to the formation and breakage of hydrogen bonds with amino acids. Finally, we found that for a high glycosylation system, glycans interact with glycoprotein and form hydrogen bonds among themselves. Moreover, the hydrogen bond profiles of a given glycan can vary when influenced by other glycans. In summary, our study provides valuable insights into the dynamics of the core region of HCV E2 glycoprotein and its associated glycans.Communicated by Ramaswamy H. Sarma.

Deciphering the molecular choreography of Janus kinase 2 inhibition via Gaussian accelerated molecular dynamics simulations: a dynamic odyssey.

The Janus kinases (JAK) are crucial targets in drug development for several diseases. However, accounting for the impact of possible structural rearrangements on the binding of different kinase inhibitors is complicated by the extensive conformational variability of their catalytic kinase domain (KD). The dynamic KD contains mainly four prominent mobile structural motifs: the phosphate-binding loop (P-loop), the αC-helix within the N-lobe, the Asp-Phe-Gly (DFG) motif, and the activation loop (A-loop) within the C-lobe. These distinct structural orientations imply a complex signal transmission path for regulating the A-loop's flexibility and conformational preference for optimal JAK function. Nevertheless, the precise dynamical features of the JAK induced by different types of inhibitors still remain elusive. We performed comparative, microsecond-long, Gaussian accelerated molecular dynamics simulations in triplicate of three phosphorylated JAK2 systems: the KD alone, type-I ATP-competitive inhibitor (CI) bound KD in the catalytically active DFG-in conformation, and the type-II inhibitor (AI) bound KD in the catalytically inactive DFG-out conformation. Our results indicate significant conformational variations observed in the A-loop and αC helix motions upon inhibitor binding. Our studies also reveal that the DFG-out inactive conformation is characterized by the closed A-loop rearrangement, open catalytic cleft of N and C-lobe, the outward movement of the αC helix, and open P-loop states. Moreover, the outward positioning of the αC helix impacts the hallmark salt bridge formation between Lys882 and Glu898 in an inactive conformation. Finally, we compared their ligand binding poses and free energy by the MM/PBSA approach. The free energy calculations suggested that the AI's binding affinity is higher than CI against JAK2 due to an increased favorable contribution from the total non-polar interactions and the involvement of the αC helix. Overall, our study provides the structural and energetic insights crucial for developing more promising type I/II JAK2 inhibitors for treating JAK-related diseases.

Microsecond dynamics of H10N7 influenza neuraminidase reveals the plasticity of loop regions and drug resistance due to the R292K mutation.

At the beginning of the last century, multiple pandemics caused by influenza (flu) viruses severely impacted public health. Despite the development of vaccinations and antiviral medications to prevent and control impending flu outbreaks, unforeseen novel strains and continuously evolving old strains continue to represent a serious threat to human life. Therefore, the recently identified H10N7, for which not much data is available for rational structure-based drug design, needs to be further explored. Here, we investigated the structural dynamics of neuraminidase N7 upon binding of inhibitors, and the drug resistance mechanisms against the oseltamivir (OTV) and laninamivir (LNV) antivirals due to the crucial R292K mutation on the N7 using the computational microscope, molecular dynamics (MD) simulations. In this study, each system underwent long 2 × 1 µs MD simulations to answer the conformational changes and drug resistance mechanisms. These long time-scale dynamics simulations and free energy landscapes demonstrated that the mutant systems showed a high degree of conformational variation compared to their wildtype (WT) counterparts, and the LNV-bound mutant exhibited an extended 150-loop conformation. Further, the molecular mechanics Poisson-Boltzmann surface area (MM/PBSA) calculation and MM/GBSA free energy decomposition were used to characterize the binding of OTV and LNV with WT, and R292K mutated N7, revealing the R292K mutation as drug-resistant, facilitated by a decline in binding interaction and a reduction in the dehydration penalty. Due to the broader binding pocket cavity of the smaller K292 mutant residue relative to the wildtype, the drug carboxylate to K292 hydrogen bonding was lost, and the area surrounding the K292 residue was more accessible to water molecules. This implies that drug resistance could be reduced by strengthening the hydrogen bond contacts between N7 inhibitors and altered N7, creating inhibitors that can form a hydrogen bond to the mutant K292, or preserving the closed cavity conformations.

Protein S-palmitoylation is markedly inhibited by 4″-alkyl ether lipophilic derivatives of EGCG, the major green tea polyphenol: In vitro and in silico studies.

S-palmitoylation is a dynamic lipid-based protein post-translational modification facilitated by a family of protein acyltransferases (PATs) commonly known as DHHC-PATs or DHHCs. It is the only lipid modification that is reversible, and this very fact uniquely qualifies it for therapeutic interventions through the development of DHHC inhibitors. Herein, we report that 4″-alkyl ether lipophilic derivatives of EGCG can effectively inhibit protein S-palmitoylation in vitro. With the help of metabolic labeling followed by copper(I)-catalyzed azide-alkyne cycloaddition Click reaction, we demonstrate that 4″-C14 EGCG and 4″-C16 EGCG markedly inhibited S-palmitoylation in various mammalian cells including HEK 293T, HeLa, and MCF-7 using both in gel fluorescence as well as confocal microscopy. Further, these EGCG derivatives were able to attenuate the S-palmitoylation to the basal level in DHHC3-overexpressed cells, suggesting that they are plausibly targeting DHHCs. Confocal microscopy data qualitatively reflected spatial and temporal distribution of S-palmitoylated proteins in different sub-cellular compartments and the inhibitory effects of 4″-C14 EGCG and 4″-C16 EGCG were clearly observed in the native cellular environment. Our findings were further substantiated by in silico analysis which revealed promising binding affinity and interactions of 4″-C14 EGCG and 4″-C16 EGCG with key amino acid residues present in the hydrophobic cleft of the DHHC20 enzyme. We also demonstrated the successful inhibition of S-palmitoylation of GAPDH by 4″-C16 EGCG. Taken together, our in vitro and in silico data strongly suggest that 4″-C14 EGCG and 4″-C16 EGCG can act as potent inhibitors for S-palmitoylation and can be employed as a complementary tool to investigate S-palmitoylation.

Elucidating the conformational dynamics of histo-blood group antigens and their interactions with the rotavirus spike protein through computational lens.

In the present study, we investigated the conformational dynamics of histo-blood group antigens (HBGAs) and their interactions with the VP8* domain of four rotavirus genotypes (P[4], P[6], P[19], and P[11]) utilizing all-atom molecular dynamics simulations in explicit water. Our study revealed distinct changes in the dynamic behavior of the same glycan due to linkage variations. We observed that LNFPI HBGA having a terminal ß linkage shows two dominant conformations after complexation, whereas only one was obtained for LNFPI with a terminal α linkage. Interestingly, both variants displayed a single dominant structure in the free state. Similarly, LNT and LNnT show a shift in their dihedral linkage profile between their two terminal monosaccharides because of a change in the linkage from ß(1-3) to ß(1-4). The molecular mechanics generalized Born surface area (MM/GBSA) calculations yielded the highest binding affinity for LNFPI(ß)/P[6] (-13.93 kcal/mol) due to the formation of numerous hydrogen bonds between VP8* and HBGAs. LNnT binds more strongly to P[11] (-12.88 kcal/mol) than LNT (-4.41 kcal/mol), suggesting a single change in the glycan linkage might impact its binding profile significantly. We have also identified critical amino acids and monosaccharides (Gal and GlcNAc) that contributed significantly to the protein-ligand binding through the per-residue decomposition of binding free energy. Moreover, we found that the interaction between the same glycan and different protein receptors within the same rotavirus genogroup influenced the micro-level dynamics of the glycan. Overall, our study helps a deeper understanding of the H-type HBGA and rotavirus spike protein interaction.Communicated by Ramaswamy H. Sarma.

Molecular level investigation for identifying potential inhibitors against thymidylate kinase of monkeypox through in silico approaches.

The need for more advanced and effective monkeypox (Mpox) treatments has become evident with numerous Mpox virus (MPXV) outbreaks. Over the years, interest has increased in developing targeted medicines that are efficient, safe, and precise while avoiding adverse effects. Here, we screened 32409 compounds against thymidylate kinase (TMPK), an emerging target for Mpox treatment. We studied their pharmacological characteristics and analyzed those through all-atom molecular dynamics simulations followed by molecular mechanics Poisson Boltzmann surface area (MM-PBSA) based free energy calculations. According to our findings, the leads CID40777874 and CID28960001 had the highest binding affinities towards TMPK with ΔGbind of -8.04 and -5.58 kcal/mol, respectively, which outperformed our control drug cidofovir (ΔGbind = -2.92 kcal/mol) in terms of binding favourability. Additionally, we observed crucial TMPK dynamics brought on by ligand-binding and identified key residues such as Phe68 and Tyr101 as the critical points of the protein-ligand interaction. The DCCM analysis revealed the role of ligand binding in stabilizing TMPK's binding region, as indicated by residual correlation motions. Moreover, the PSN analysis revealed that the interaction with ligand induces changes in residual network properties, enhancing the stability of complexes. We successfully identified novel compounds that may serve as potential building blocks for constructing contemporary antivirals against MPXV and highlighted the molecular mechanisms underlying their binding with TMPK. Overall, our findings will play a significant role in advancing the development of new therapies against Mpox and facilitating a comprehensive understanding of their interaction patterns.Communicated by Ramaswamy H. Sarma.

Computer-aided Affinity Enhancement of a Cross-reactive Antibody against Dengue Virus Envelope Domain III.

The dengue virus (DENV), composed of four distinct but serologically related Flaviviruses, causes the most important emerging viral disease, with nearly 400 million infections yearly. Currently, there are no approved therapies. Although DENV infection induces lifelong immunity against the same serotype, the antibodies raised contribute to severe disease in heterotypic infections. Therefore, understanding the mechanism of DENV neutralization by antibodies is crucial in the design of vaccines against all serotypes. This study reports a comparative structural and energetic analysis of the monoclonal antibody (mAb) 4E11 in complex with its target domain III of the envelope protein for all four DENV serotypes. We use extensive replica molecular dynamics simulations in conjunction with the binding free energy calculations. Further single point and double mutations were designed through computational site-directed mutagenesis and observed that the re-engineered antibody exhibits high affinity to binding and broadly neutralizing activity against serotypes. Our results showed improved binding affinity by the gain of enthalpy, which could be attributed to the stabilization of salt-bridge and hydrogen bond interactions at the antigen-antibody interface. The findings provide valuable results in understanding the structural dynamics and energetic contributions that will be helpful to the design of high-affinity antibodies against dengue infections.

Structural analysis of human ATE1 isoforms and their interactions with Arg-tRNAArg.

Posttranslational protein arginylation has been shown as a key regulator of cellular processes in eukaryotes by affecting protein stability, function, and interaction with macromolecules. Thus, the enzyme Arginyltransferase and its targets, are of immense interest to modulate cellular processes in the normal and diseased state. While the study on the effect of this posttranslational modification in mammalian systems gained momentum in the recent times, the detail structures of human ATE1 (hATE1) enzymes has not been investigated so far. Thus, the purpose of this study was to predict the overall structure and the structure function relationship of hATE1 enzyme and its four isoforms. The structure of four ATE1 isoforms were modelled and were docked with 3'end of the Arg-tRNAArg which acts as arginine donor in the arginylation reaction, followed by MD simulation. All the isoforms showed two distinct domains. A compact domain and a somewhat flexible domain as observed in the RMSF plot. A distinct similarity in the overall structure and interacting residues were observed between hATE1-1 and X4 compared to hATE1-2 and 5. While the putative active sites of all the hATE1 isoforms were located at the same pocket, differences were observed in the active site residues across hATE1 isoforms suggesting different substrate specificity. Mining of nsSNPs showed several nsSNPs including cancer associated SNPs with deleterious consequences on hATE1 structure and function. Thus, the current study for the first time shows the structural differences in the mammalian ATE1 isoforms and their possible implications in the function of these proteins.Communicated by Ramaswamy H. Sarma.

Conformational preferences of triantennary and tetraantennary hybrid N-glycans in aqueous solution: Insights from 20 µs long atomistic molecular dynamic simulations.

In the current study, we have investigated the conformational dynamics of a triantennary (N-glycan1) and tetraantennary (N-glycan2) hybrid N-glycans found on the surface of the HIV glycoprotein using 20 µs long all-atom molecular dynamics (MD) simulations. The main objective of the present study is to elucidate the influence of adding a complex branch on the overall glycan structural dynamics. Our investigation suggests that the average RMSD value increases when a complex branch is added to N-glycan1. However, the RMSD distribution is relatively wider in the case of N-glycan1 compared to N-glycan2, which indicates that multiple complex branches restrict the conformational variability of glycans. A similar observation is obtained from the principal component analysis of both glycans. All the puckering states (4C1 to 1C4) of each monosaccharide except mannose are sampled in our simulations, although the 4C1 chair form is energetically more favorable than 1C4. In N-glycan1, the 1-6 linkage in the mannose branch [Man(9)-α(1-6)-Man(5)] stays in the gauche-gauche cluster, whereas it moves towards trans-gauche in N-glycan2. For both glycans, mannose branches are more flexible than the complex branches, and adding a complex branch does not influence the dynamics of the mannose branches. We have noticed that the end-to-end distance of the complex branch shortens by ∼ 10 Å in the presence of another complex branch. This suggests that in the presence of an additional complex branch, the other complex branch adopts a close folded structure. All these conformational changes involve the selective formation of inter-residue and water-mediated hydrogen-bond networks.Communicated by Ramaswamy H. Sarma.

Comparison of the conformational dynamics of an N-glycan in implicit and explicit solvents.

All-atom MD simulations provide the atomic details of glycan structure in solution, but extensive sampling is required for simulating the transition between rotameric states. For carbohydrate modeling, the motions of the glycosidic linkages in a glycan are often sampled well on the sub-µs time scale. Consequently, simulations of large glycans or glycoconjugates in aqueous environments are very challenging at the atomistic level because of a huge number of water molecules, which eventually slows down the conformational sampling. An alternative to the all-atom approach is the multiscale simulation, where the glycan is in all-atom form while the solvent is treated implicitly. Here we present a comparative analysis of the implicit model with the currently used explicit model using Gaussian accelerated molecular dynamics (GaMD). Here we used a hybrid N-glycan to investigate the accuracy of the implicit solvent model. Estimating several structural parameters, namely dihedral torsional angles, puckering angles, and end-to-end distances, yields a complete classification of structural space in both solvent models. Both solvent models displayed similar conformations at the monosaccharide level, whereas minor differences were observed in the global conformation. The only major difference was observed in the inter-residue hydrogen bonds, which increased by 2-fold in the explicit solvent model. Detailed analysis of structural parameters established a balanced trade-off between the computational speed and accuracy for studying long-chain glycan molecules in an implicit solvent.

Finding potent inhibitors against SARS-CoV-2 main protease through virtual screening, ADMET, and molecular dynamics simulation studies.

Currently, no antiviral drug or vaccine is available to treat COVID-19 caused by SARS-CoV-2. This underscores an urgent need for developing a drug against SARS-CoV-2. The main protease (3CLpro) of SARS-CoV-2 is considered an essential protein for maintaining the viral life cycle and, therefore, a potential target for drug development. In a recent study, 1000 potential ligands were identified for 3CLpro by screening 1.3 billion compounds from the ZINC15 library. In the current study, we have further screened these 1000 compounds using structure-based virtual screening utilizing the Schrödinger suite and identified nine compounds having a docking score of ∼ -11.0 kcal/mol or less. The top 5 hits display good pharmacological profiles revealing better absorption, proper permeability across the membrane, uniform distribution, and non-toxic. The molecular docking study is further complemented by molecular dynamics simulations of the top 5 docked complexes. The binding free energy analyses via the molecular mechanics generalized Born surface area (MM/GBSA) scheme reveals that ZINC000452260308 is the most potent (ΔGbind = -14.31 kcal/mol) inhibitor. The intermolecular van der Waals interactions mainly drive the 3CLpro-ligand association. This new compound may have great potential as a lead molecule to develop a new antiviral drug to fight against COVID-19.Communicated by Ramaswamy H. Sarma.

Investigating the mechanism of recognition and structural dynamics of nucleoprotein-RNA complex from Peste des petits ruminants virus via Gaussian accelerated molecular dynamics simulations.

The nucleocapsid (N) protein from Peste des petits ruminants virus enwraps the nascent genomic RNA primarily to protect it from cellular enzymatic degradation. Here, we have combined molecular modeling and 1 µs long Gaussian accelerated molecular dynamics simulations to study the structural and conformational properties of the apo N-protein and three protein-RNA complexes, namely wild type (WT), MT1 (I46T/E297D/G342E), and MT2 (I46T/E297D/G342E/W333G). The root-mean-squared deviation (RMSD) analysis reveals that although MT2 deviates most significantly from the initial structure, the RNA binding region is more stable compared to WT or MT1. Further, the flexible nature of the N protein is revealed from the principal component analysis. Our study shows that the solvent accessible surface area of the binding region increases drastically for both mutant complexes compared to WT. The dynamic cross-correlation analysis suggests that the overall anticorrelated motion weakens after the RNA binding. MT2 displays a relatively larger positive correlation than WT or MT1. The binding free energy is estimated for all three complexes via the molecular mechanics/generalized Born surface (MM/GBSA) scheme, and it decreases in the order WT > MT1 > MT2. In all cases, the protein-RNA binding is mainly driven by the van der Waals interactions since the intermolecular electrostatic interaction is overcompensated by desolvation energy. All hotspot residues are identified from the per-residue decomposition of the total binding free energy. In MT2, RNA contributes most favorably compared to WT or MT1. We believe our study will help in understanding the mechanism of RNA recognition by N proteins.Communicated by Ramaswamy H. Sarma.

Unraveling the Molecular Mechanism of Recognition of Human Interferon-Stimulated Gene Product 15 by Coronavirus Papain-Like Proteases: A Multiscale Simulation Study.

The papain-like protease (PLpro) of the coronavirus (CoV) family plays an essential role in processing the viral polyprotein and immune evasion. Additional proteolytic activities of PLpro include deubiquitination and deISGylation, which can reverse the post-translational modification of cellular proteins conjugated with ubiquitin or (Ub) or Ub-like interferon-stimulated gene product 15 (ISG15). These activities regulate innate immune responses against viral infection. Thus, PLpro is a potential antiviral target. Here, we have described the structural and energetic basis of recognition of PLpro by the human ISG15 protein (hISG15) using atomistic molecular dynamics simulation across the CoV family, i.e., MERS-CoV (MCoV), SARS-CoV (SCoV), and SARS-CoV-2 (SCoV2). The cumulative simulation length for all trajectories was 32.0 µs. In the absence of the complete crystal structure of complexes, protein-protein docking was used. A mutation (R167E) was introduced across all three PLpro to study the effect of mutation on the protein-protein binding. Our study reveals that the apo-ISG15 protein remains closed while it adopts an open conformation when bound to PLpro, although the degree of openness varies across the CoV family. The binding free energy analysis suggests that hISG15 binds more strongly with SCoV2-PLpro compared to SCoV or MCoV. The intermolecular electrostatic interaction drives the hISG15-PLpro complexation. Our study showed that SCoV or MCoV-PLpro binds more strongly with the C-domain of hISG15, while SCoV2-PLpro binds more favorably the N-domain of hISG15. Overall, our study explains the molecular basis of differential deISGylating activities of PLpro among the CoV family and the specificity of SCoV2-PLpro toward hISG15.

Elucidating biophysical basis of binding of inhibitors to SARS-CoV-2 main protease by using molecular dynamics simulations and free energy calculations.

The recent outbreak of novel "coronavirus disease 2019" (COVID-19) has spread rapidly worldwide, causing a global pandemic. In the present work, we have elucidated the mechanism of binding of two inhibitors, namely α-ketoamide and Z31792168, to SARS-CoV-2 main protease (Mpro or 3CLpro) by using all-atom molecular dynamics simulations and free energy calculations. We calculated the total binding free energy (ΔGbind) of both inhibitors and further decomposed ΔGbind into various forces governing the complex formation using the Molecular Mechanics/Poisson-Boltzmann Surface Area (MM/PBSA) method. Our calculations reveal that α-ketoamide is more potent (ΔGbind= - 9.05 kcal/mol) compared to Z31792168 (ΔGbind= - 3.25 kcal/mol) against COVID-19 3CLpro. The increase in ΔGbind for α-ketoamide relative to Z31792168 arises due to an increase in the favorable electrostatic and van der Waals interactions between the inhibitor and 3CLpro. Further, we have identified important residues controlling the 3CLpro-ligand binding from per-residue based decomposition of the binding free energy. Finally, we have compared ΔGbind of these two inhibitors with the anti-HIV retroviral drugs, such as lopinavir and darunavir. It is observed that α-ketoamide is more potent compared to lopinavir and darunavir. In the case of lopinavir, a decrease in van der Waals interactions is responsible for the lower binding affinity compared to α-ketoamide. On the other hand, in the case of darunavir, a decrease in the favorable intermolecular electrostatic and van der Waals interactions contributes to lower affinity compared to α-ketoamide. Our study might help in designing rational anti-coronaviral drugs targeting the SARS-CoV-2 main protease.Communicated by Ramaswamy H. Sarma.

Computational Investigation of Structural Dynamics of SARS-CoV-2 Methyltransferase-Stimulatory Factor Heterodimer nsp16/nsp10 Bound to the Cofactor SAM.

Recently, a highly contagious novel coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, has emerged, posing a global threat to public health. Identifying a potential target and developing vaccines or antiviral drugs is an urgent demand in the absence of approved therapeutic agents. The 5'-capping mechanism of eukaryotic mRNA and some viruses such as coronaviruses (CoVs) are essential for maintaining the RNA stability and protein translation in the virus. SARS-CoV-2 encodes S-adenosyl-L-methionine (SAM) dependent methyltransferase (MTase) enzyme characterized by nsp16 (2'-O-MTase) for generating the capped structure. The present study highlights the binding mechanism of nsp16 and nsp10 to identify the role of nsp10 in MTase activity. Furthermore, we investigated the conformational dynamics and energetics behind the binding of SAM to nsp16 and nsp16/nsp10 heterodimer by employing molecular dynamics simulations in conjunction with the Molecular Mechanics Poisson-Boltzmann Surface Area (MM/PBSA) method. We observed from our simulations that the presence of nsp10 increases the favorable van der Waals and electrostatic interactions between SAM and nsp16. Thus, nsp10 acts as a stimulator for the strong binding of SAM to nsp16. The hydrophobic interactions were predominately identified for the nsp16-nsp10 interactions. Also, the stable hydrogen bonds between Ala83 (nsp16) and Tyr96 (nsp10), and between Gln87 (nsp16) and Leu45 (nsp10) play a vital role in the dimerization of nsp16 and nsp10. Besides, Computational Alanine Scanning (CAS) mutagenesis was performed, which revealed hotspot mutants, namely I40A, V104A, and R86A for the dimer association. Hence, the dimer interface of nsp16/nsp10 could also be a potential target in retarding the 2'-O-MTase activity in SARS-CoV-2. Overall, our study provides a comprehensive understanding of the dynamic and thermodynamic process of binding nsp16 and nsp10 that will contribute to the novel design of peptide inhibitors based on nsp16.

Structural changes in DNA-binding proteins on complexation.

Characterization and prediction of the DNA-biding regions in proteins are essential for our understanding of how proteins recognize/bind DNA. We analyze the unbound (U) and the bound (B) forms of proteins from the protein-DNA docking benchmark that contains 66 binary protein-DNA complexes along with their unbound counterparts. Proteins binding DNA undergo greater structural changes on complexation (in particular, those in the enzyme category) than those involved in protein-protein interactions (PPI). While interface atoms involved in PPI exhibit an increase in their solvent-accessible surface area (ASA) in the bound form in the majority of the cases compared to the unbound interface, protein-DNA interactions indicate increase and decrease in equal measure. In 25% structures, the U form has missing residues which are located in the interface in the B form. The missing atoms contribute more toward the buried surface area compared to other interface atoms. Lys, Gly and Arg are prominent in disordered segments that get ordered in the interface on complexation. In going from U to B, there may be an increase in coil and helical content at the expense of turns and strands. Consideration of flexibility cannot distinguish the interface residues from the surface residues in the U form.