The geometries of interacting arginine-carboxyls in proteins

The geometries are reported for interacting arginine-carboxyl pairs obtained from 37 high resolution protein structures solved to a resolution of 2.0 A or better. The closest interatomic distance between the guanidinium and carboxyl is less than 4.2 A for 74 arginine and carboxyl groups, with the majority of these lying within hydrogen-bonding distance (2.6-3.0 A). Interacting pairs have been transformed into a common orientation, and arginine-carboxyl, and carboxyl-arginine geometries have been calculated. This has been defined in terms of the spherical polar angles T theta, T phi, and the angle P, between the guanidinium and carboxyl planes. Results show a clear preference for the guanidinium and carboxyl groups to be approximately coplanar, and for the carboxyl oxygens to hydrogen bond with the guanidinium nitrogens. Single nitrogen-single oxygen is the most common type of interaction, however twin nitrogen-twin oxygen interactions also occur frequently. The majority of these occur between the carboxyl oxygens and the NH1 and NE atoms of the arginine, and are only rarely observed for NH1 and NH2. The information presented may be of use in the modelling of arginine-carboxyl interactions within proteins.     

An autoantibody to single-stranded DNA: comparison of the three-dimensional structures of the unliganded Fab and a deoxynucleotide-Fab complex.

Crystal structures of the Fabs from an autoantibody (BV04-01) with specificity for single-stranded DNA have been determined in the presence and absence of a trinucleotide of deoxythymidylic acid, d(pT)3. Formation of the ligand-protein complex was accompanied by small adjustments in the orientations of the variable (VL and VH) domains. In addition, there were local conformational changes in the first hypervariable loop of the light chain and the third hypervariable loop of the heavy chain, which together with the domain shifts led to an improvement in the complementarity of nucleotide and Fab. The sugar-phosphate chain adopted an extended and "open" conformation, with the base, sugar, and phosphate components available for interactions with the protein. Nucleotide 1 (5'-end) was associated exclusively with the heavy chain, nucleotide 2 was shared by both heavy and light chains, and nucleotide 3 was bound by the light chain. The orientation of phosphate 1 was stabilized by hydrogen bonds with serine H52a and asparagine H53. Phosphate 2 formed an ion pair with arginine H52, but no other charge-charge interactions were observed. Insertion of the side chain of histidine L27d between nucleotides 2 and 3 resulted in a bend in the sugar-phosphate chain. The most dominant contacts with the protein involved the central thymine base, which was immobilized by cooperative stacking and hydrogen bonding interactions. This base was intercalated between a tryptophan ring (no. H100a) from the heavy chain and a tyrosine ring (no. L32) from the light chain. The resulting orientation of thymine was favorable for the simultaneous formation of two hydrogen bonds with the backbone carbonyl oxygen and the side chain hydroxyl group of serine L91 (the thymine atoms were the hydrogen on nitrogen 3 and keto oxygen 4).     

Specific arginine mediated RNA recognition

In many biological systems substantial roles are played by interactions between amino acids and RNA. Among amino acids L-arginine seems to be particularly relevant, because the guanidinium group of arginine side chain can potentially form five hydrogen bonds with appropriately positioned acceptor groups of RNA. Extensive studies reveal that specific arginine recognition is achieved by many different RNAs over a broad range of binding affinities. Arginine is frequently found among amino acids in the nucleic acid-binding motifs in various proteins. For example, specific binding of the HIV-1 Tat protein to its RNA site (TAR) is mediated by a single arginine residue. Free arginine can be also bound by the guanosine site in the group I Tetrahymena ribosomal RNA intron catalytic centre, as well as by numerous RNA motifs, called arginine aptamers, which have been selected in vitro.     

Ion pair formation of phosphorylated amino acids and lysine and arginine side chains: A theoretical study

Protein phosphorylation is one of the major signal transduction mechanisms for controlling and regulating intracellular processes. Phosphorylation of specific hydroxylated amino acid side chains (Ser, Thr, Tyr) by protein kinases can activate numerous enzymes; this effect can be reversed by the action of protein phosphatases. Here we report ab initio (HF/6-31G* and Becke3LYP/6-31G*) and semiempirical (PM3) molecular orbital calculations pertinent to the ion pair formation of the phosphorylated amino acids with the basic side chains of Lys and Arg. Methyl-, ethyl-, and phenylphosphate, as well as methylamine and methylguanidinium were used as model compounds for the phosphorylated and basic amino acids, respectively. Phosphorylated amino acids were calculated as mono- and divalent anions. Our results indicate that the PSer/PThr ion pair interaction energies are stronger than those with PTyr. Moreover, the interaction energies with the amino group of Lys are generally more favorable than with the guanidinium group of Arg. The Lys amino groups form stable bifurcated hydrogen bonded structures; while the Arg guanidinium group can form a bidentate hydrogen bonded structure. Reasonable values for the interaction free energies in aqueous solution were obtained for some complexes by the inclusion of a solvent reaction field in the computation (PM3-SM3).     

Refined crystal structure of cytoplasmic malate dehydrogenase at 2.5-A resolution

The molecular structure of cytoplasmic malate dehydrogenase from pig heart has been refined by alternating rounds of restrained least-squares methods and model readjustment on an interactive graphics system. The resulting structure contains 333 amino acids in each of the two subunits, 2 NAD molecules, 471 solvent molecules, and 2 large noncovalently bound molecules that are assumed to be sulfate ions. The crystallographic study was done on one entire dimer without symmetry restraints. Analysis of the relative position of the two subunits shows that the dimer does not obey exact 2-fold rotational symmetry; instead, the subunits are related by a 173 degrees rotation. The structure results in a R factor of 16.7% for diffraction data between 6.0 and 2.5 A, and the rms deviations from ideal bond lengths and angles are 0.017 A and 2.57 degrees, respectively. The bound coenzyme in addition to hydrophobic interactions makes numerous hydrogen bonds that either are directly between NAD and the enzyme or are with solvent molecules, some of which in turn are hydrogen bonded to the enzyme. The carboxamide group of NAD is hydrogen bonded to the side chain of Asn-130 and via a water molecule to the backbone nitrogens of Leu-157 and Asp-158 and to the carbonyl oxygen of Leu-154. Asn-130 is one of the corner residues in a beta-turn that contains the lone cis peptide bond in cytoplasmic malate dehydrogenase, situated between Asn-130 and Pro-131. The active site histidine, His-186, is hydrogen bonded from nitrogen ND1 to the carboxylate of Asp-158 and from its nitrogen NE2 to the sulfate ion bound in the putative substrate binding site. In addition to interacting with the active site histidine, this sulfate ion is also hydrogen bonded to the guanidinium group of Arg-161, to the carboxamide group of Asn-140, and to the hydroxyl group of Ser-241. It is speculated that the substrate, malate or oxaloacetate, is bound in the sulfate binding site with the substrate 1-carboxyl hydrogen bonded to the guanidinium group of Arg-161.     

Voltage-sensing arginines in a potassium channel permeate and occlude cation-selective pores

Voltage-gated ion channels sense voltage by shuttling arginine residues located in the S4 segment across the membrane electric field. The molecular pathway for this arginine permeation is not understood, nor is the filtering mechanism that permits passage of charged arginines but excludes solution ions. We find that substituting the first S4 arginine with smaller amino acids opens a high-conductance pathway for solution cations in the Shaker K(+) channel at rest. The cationic current does not flow through the central K(+) pore and is influenced by mutation of a conserved residue in S2, suggesting that it flows through a protein pathway within the voltage-sensing domain. The current can be carried by guanidinium ions, suggesting that this is the pathway for transmembrane arginine permeation. We propose that when S4 moves it ratchets between conformations in which one arginine after another occupies and occludes to ions the narrowest part of this pathway.     

Interaction of arginine,lysine, and guanidine with surface residues of lysozyme: implication to protein stability

Additives are widely used to suppress aggregation of therapeutic proteins. However, the molecular mechanisms of effect of additives to stabilize proteins are still unclear. To understand this, we herein perform molecular dynamics simulations of lysozyme in the presence of three commonly used additives: arginine, lysine, and guanidine. These additives have different effects on stability of proteins and have different structures with some similarities; arginine and lysine have aliphatic side chain, while arginine has a guanidinium group. We analyze atomic contact frequencies to study the interactions of the additives with individual residues of lysozyme. Contact coefficient, quantified from contact frequencies, is helpful in analyzing the interactions with the guanidine groups as well as aliphatic side chains of arginine and lysine. Strong preference for contacts to the additives (over water) is seen for the acidic followed by polar and the aromatic residues. Further analysis suggests that the hydration layer around the protein surface is depleted more in the presence of arginine, followed by lysine and guanidine. Molecular dynamics simulations also reveal that the internal dynamics of protein, as indicated by the lifetimes of the hydrogen bonds within the protein, changes depending on the additives. Particularly, we note that the side-chain hydrogen-bonding patterns within the protein differ with the additives, with several side-chain hydrogen bonds missing in the presence of guanidine. These results collectively indicate that the aliphatic chain of arginine and lysine plays a critical role in the stabilization of the protein.     

The probable structure of the protamine-DNA complex

A detailed molecular structure is proposed for the human protamine-DNA complex, which has hitherto been largely a mystery. The structure was created with virtual modeling software (AmiraMol), employing logical deduction as the primary investigative tool. A beta-sheet structure for the protein component is essentially mandated, as the alternatives can be decisively excluded. A dimeric structure too is essentially mandated, since the cysteine residues of protamines P1 and P2 are invariably aligned in all species having both chains. The cross-sectional and axial spacings of arginine guanidinium groups in this protein structure can be perfectly aligned with those of phosphate groups in DNA according to the DNA structure proposed by Wu. This is a non-helical structure, whose possible occurrence in certain plasmids has been suggested by experimental observations. The unit cell of this protamine-DNA complex is essentially devoid of steric hindrances, and heavily favored by a multitude of ionic and hydrogen bonds. The packing of adjacent "unit cells" of the protamine-DNA structure is based on a complex array of salt bridges, the mere existence of which is so fortuitous that it is virtually inconceivable that it comes about through a mere modeling "coincidence". The possible significance of the structure beyond the sperm cell is discussed.     

A study on the effect of surface lysine to arginine mutagenesis on protein stability and structure using green fluorescent protein

Two positively charged basic amino acids, arginine and lysine, are mostly exposed to protein surface, and play important roles in protein stability by forming electrostatic interactions. In particular, the guanidinium group of arginine allows interactions in three possible directions, which enables arginine to form a larger number of electrostatic interactions compared to lysine. The higher pKa of the basic residue in arginine may also generate more stable ionic interactions than lysine. This paper reports an investigation whether the advantageous properties of arginine over lysine can be utilized to enhance protein stability. A variant of green fluorescent protein (GFP) was created by mutating the maximum possible number of lysine residues on the surface to arginines while retaining the activity. When the stability of the variant was examined under a range of denaturing conditions, the variant was relatively more stable compared to control GFP in the presence of chemical denaturants such as urea, alkaline pH and ionic detergents, but the thermal stability of the protein was not changed. The modeled structure of the variant indicated putative new salt bridges and hydrogen bond interactions that help improve the rigidity of the protein against different chemical denaturants. Structural analyses of the electrostatic interactions also confirmed that the geometric properties of the guanidinium group in arginine had such effects. On the other hand, the altered electrostatic interactions induced by the mutagenesis of surface lysines to arginines adversely affected protein folding, which decreased the productivity of the functional form of the variant. These results suggest that the surface lysine mutagenesis to arginines can be considered one of the parameters in protein stability engineering.     

Mechanistic insights from the structures of HincII bound to cognate DNA cleaved from addition of Mg2+ and Mn2+

The three-dimensional X-ray crystal structures of HincII bound to cognate DNA containing GTCGAC and Mn(2+) or Mg(2+), at 2.50A and 2.95A resolution, respectively, are presented. In both structures, the DNA is found cleaved, and the positions of the active-site groups, cleaved phosphate group, and 3' oxygen atom of the leaving group are in very similar positions. Two highly occupied Mn(2+) positions are found in each active site of the four crystallographically independent subunit copies in the HincII/DNA/Mn(2+) structure. The manganese ion closest to the previously identified single Ca(2+) position of HincII is shifted 1.7A and has lost direct ligation to the active-site aspartate residue, Asp127. A Mn(2+)-ligated water molecule in a position analogous to that seen in the HincII/DNA/Ca(2+) structure, and proposed to be the attacking nucleophile, is beyond hydrogen bonding distance from the active-site lysine residue, Lys129, but remains within hydrogen bonding distance from the proRp oxygen atom of the phosphate group 3' to the scissile phosphate group. In addition, the position of the cleaved phosphate group is on the opposite side of the axis connecting the two metal ions relative to that found in the BamHI/product DNA/Mn(2+) structure. Mechanistic implications are discussed, and a model for the two-metal-ion mechanism of DNA cleavage by HincII is proposed.     

A Look at Arginine in Membranes

Here, we review the current knowledge about the energetics of arginine insertion into the bilayer hydrocarbon core, and we discuss discrepancies between experimental and computational studies of the insertion process. While simulations suggest that it should be very costly to place arginine into the hydrocarbon core, experiments show that arginine is found there. Both types of studies suggest that arginine insertion into the bilayer involves substantial bilayer deformation, with multiple hydrogen bonds between the arginine guanidinium group and lipid polar groups. It is possible that the discrepancies concerning the insertion cost of arginine arise because simulations overestimate the cost associated with bilayer deformation and underestimate the ability of the bilayer to adapt to charged and polar groups. This is an active area of research, and there is no doubt that a consensus view of arginine in membranes will soon emerge.     

Cation-pi interactions in protein-protein interfaces

Arginine is an abundant residue in protein-protein interfaces. The importance of this residue relates to the versatility of its side chain in intermolecular interactions. Different classes of protein-protein interfaces were surveyed for cation-pi interactions. Approximately half of the protein complexes and one-third of the homodimers analyzed were found to contain at least one intermolecular cation-pi pair. Interactions between arginine and tyrosine were found to be the most abundant. The electrostatic interaction energy was calculated to be approximately 3 kcal/mol, on average. A distance-based search of guanidinium:aromatic interactions was also performed using the Macromolecular Structure Database (MSD). This search revealed that half of the guanidinium:aromatic pairs pack in a coplanar manner. Furthermore, it was found that the cationic group of the cation-pi pair is frequently involved in intermolecular hydrogen bonds. In this manner the arginine side chain can participate in multiple interactions, providing a mechanism for inter-protein specificity. Thus, the cation-pi interaction is established as an important contributor to protein-protein interfaces.     

Identification of two novel arginine binding DNAs.

RNA tertiary structure is known to play critical roles in RNA-protein recognition and RNA function. To examine how DNA tertiary structure might relate to RNA structure, we performed in vitro selection experiments to identify single-stranded DNAs that specifically bind arginine, and compared the results with analogous experiments performed with RNA. In the case of RNA, a motif related to the arginine binding site in human immunodeficiency virus TAR RNA was commonly found, whereas in the case of DNA, two novel motifs and no TAR-like structures were found. One DNA motif, found in approximately 40% of the cloned sequences, forms of hairpin structure with a highly conserved 10 nucleotide loop, whereas the second motif is especially rich in G residues. Chemical interference and mutagenesis experiments identified nucleotides in both motifs that form specific arginine binding sites, and dimethylsulfate footprinting experiments identified single guanine residues in both that are protected from methylation in the presence of arginine, suggesting possible sites of arginine contact or conformational changes in the DNAs. Circular dichroism experiments indicated that both DNAs undergo conformational changes upon arginine binding and that the arginine guanidinium group alone is responsible for binding. A model for the G-rich motif is proposed in which mixed guanine and adenine quartets may form a novel DNA structure. Arginine binding DNAs and RNAs should provide useful model systems for studying nucleic acid tertiary structure.     

Interactions between l‐arginine/l‐arginine derivatives and lysozyme and implications to their inhibition effects on protein aggregation

l ‐Arginine (Arg), l ‐homoarginine (HArg), l ‐arginine ethylester (ArgEE), and l ‐arginine methylester (ArgME) were found effective in inhibiting protein aggregation, but the molecular mechanisms are not clear. Herein, stopped‐flow fluorescence spectroscopy, isothermal titration calorimetry, and mass spectroscopy were used to investigate the folding kinetics of lysozyme and the interactions of the additives with lysozyme. It was found that the interactions of ArgME and ArgEE with lysozyme were similar to that of guanidine hydrochloride and were much stronger than those of Arg and HArg. The binding forces were all mainly hydrogen bonding and cation‐π interaction from the guanidinium group, but their differences in molecular states led to the significantly different binding strengths. The additives formed molecular clusters in an increasing order of ArgEE, ArgME, HArg, and Arg. Arg and HArg mainly formed annular clusters with head‐to‐tail bonding, while ArgME and ArgEE formed linear clusters with guanidinium groups stacking. The interactions between the additives and lysozyme were positively related to the monomer contents. That is, the monomers were the primary species that participated in the direct interactions due to their intact guanidinium groups and small sizes, while the clusters performed as barriers to crowd out the protein–protein interactions for aggregation. Thus, it is concluded that the effects of Arg and its derivatives on protein aggregation stemmed from the direct interactions by the monomers and the crowding effects by the clusters. Interplay of the two effects led to the differences in their inhibition effects on protein aggregation. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1316–1324, 2013     

Effects of nucleotide bromination on the stabilities of Z-RNA and Z-DNA: a molecular mechanics/thermodynamic perturbation study

The structures of ZI- and ZII-form RNA and DNA oligonucleotides were energy minimized in vacuum using the AMBER molecular mechanics force field. Alternating C-G sequences were studied containing either unmodified nucleotides, 8-bromoguanosine in place of all guanosine residues, 5-bromocytidine in place of all cytidine residues, or all modified residues. Some molecules were also energy minimized in the presence of H2O and cations. Free energy perturbation calculations were done in which G8 and C5 hydrogen atoms in one or two residues of Z-form RNAs and DNAs were replaced in a stepwise manner by bromines. Bromination had little effect on the structures of the energy-minimized molecules. Both the minimized molecular energies and the results of the perturbation calculations indicate that bromination of guanosine at C8 will stabilize the Z forms of RNA and DNA relative to the nonbrominated Z form, while bromination of cytidine at C5 stabilizes Z-DNA and destabilizes Z-RNA. These results are in agreement with experimental data. The destabilizing effect of br5C in Z-RNAs is apparently due to an unfavorable interaction between the negatively charged C5 bromine atom and the guanosine hydroxyl group. The vacuum-minimized energies of the ZII-form oligonucleotides are lower than those of the corresponding ZI-form molecules for both RNA and DNA. Previous x-ray diffraction, nmr, and molecular mechanics studies indicate that hydration effects may favor the ZI conformation over the ZII form in DNA. Molecular mechanics calculations show that the ZII-ZI energy differences for the RNAs are greater than three times those obtained for the DNAs. This is due to structurally reinforcing hydrogen-bonding interactions involving the hydroxyl groups in the ZII form, especially between the guanosine hydroxyl hydrogen atom and the 3'-adjacent phosphate oxygen. In addition, the cytidine hydroxyl oxygen forms a hydrogen bond with the 5'-adjacent guanosine amino group in the ZII-form molecule. Both of these interactions are less likely in the ZI-form molecule: the former due to the orientation of the GpC phosphate away from the guanosine ribose in the ZI form, and the latter apparently due to competitive hydrogen bonding of the cytidine 2'-hydroxyl hydrogen with the cytosine carbonyl oxygen in the ZI form. The hydrogen-bonding interaction between the cytidine hydroxyl oxygen and the 5'-adjacent guanosine amino group in Z-RNA twists the amino group out of the plane of the base. This may be responsible for differences in the CD and Raman spectra of Z-RNA and Z-DNA.     

MutH complexed with hemi- and unmethylated DNAs: coupling base recognition and DNA cleavage

MutH initiates mismatch repair by nicking the transiently unmethylated daughter strand 5' to a GATC sequence. Here, we report crystal structures of MutH complexed with hemimethylated and unmethylated GATC substrates. Both structures contain two Ca2+ ions jointly coordinated by a conserved aspartate and the scissile phosphate, as observed in the restriction endonucleases BamHI and BglI. In the hemimethylated complexes, the active site is more compact and DNA cleavage is more efficient. The Lys residue in the conserved DEK motif coordinates the nucleophilic water in conjunction with the phosphate 3' to the scissile bond; the same Lys is also hydrogen bonded with a carbonyl oxygen in the DNA binding module. We propose that this Lys, which is conserved in many restriction endonucleases and is replaced by Glu or Gln in BamHI and BglII, is a sensor for DNA binding and the linchpin that couples base recognition and DNA cleavage.     

Why nature chose phosphate to modify proteins

The advantageous chemical properties of the phosphate ester linkage were exploited early in evolution to generate the phosphate diester linkages that join neighbouring bases in RNA and DNA (Westheimer 1987 Science 235, 1173-1178). Following the fixation of the genetic code, another use for phosphate ester modification was found, namely reversible phosphorylation of the three hydroxyamino acids, serine, threonine and tyrosine, in proteins. During the course of evolution, phosphorylation emerged as one of the most prominent types of post-translational modification, because of its versatility and ready reversibility. Phosphoamino acids generated by protein phosphorylation act as new chemical entities that do not resemble any natural amino acid, and thereby provide a means of diversifying the chemical nature of protein surfaces. A protein-linked phosphate group can form hydrogen bonds or salt bridges either intra- or intermolecularly, creating stronger hydrogen bonds with arginine than either aspartate or glutamate. The unique size of the ionic shell and charge properties of covalently attached phosphate allow specific and inducible recognition of phosphoproteins by phosphospecific-binding domains in other proteins, thus promoting inducible protein-protein interaction. In this manner, phosphorylation serves as a switch that allows signal transduction networks to transmit signals in response to extracellular stimuli.     

Mechanism of charge compensation and impairment of co-operative functions in haemoglobin Tacoma (Arg B12(30)beta leads to Ser).

A difference Fourier synthesis of deoxyhaemoglobin Tacoma minus deoxyhaemoglobin A at 3.5 Å resolution has been calculated. The map shows a large negative peak due to the removal of the guanidinium group of Arg B12(30)β, surrounded by positive and negative peaks indicative of some atoms moving towards, and others away from, the vacated site. Among the latter, the most important is the carboxylate of Glu B8(26)β which is hydrogen-bonded to the guanidinium group of the arginine in haemoglobin A, but swings round its α-β carbon bond towards the imidazoles of histidines G18(116) and 19(117)β in haemoglobin Tacoma. This movement would raise the pK values of the histidines, so that their positive charges compensated for the loss of the arginine. This may explain why haemoglobin Tacoma has the same electrophoretic mobility as haemoglobin A. It is shown that haemoglobin Tacoma has a lower oxygen equilibrium constant K_T and a larger allosteric constant L than haemoglobin A. The lowering of K_T may be due to the loosening of the T structure in haemoglobin Tacoma consequent upon the removal of the hydrogen bonds made by the guanidinium group of Arg B12(30)β at the α₁β₁ contact. Their removal also accounts for the decreased stability of haemoglobin Tacoma. We cannot yet explain its diminished Bohr effect, nor the increase in L.     

Sequence specific DNA binding of Ets-1 transcription factor: molecular dynamics study on the Ets domain--DNA complexes

Molecular dynamics (MD) simulations for Ets-1 ETS domain-DNA complexes were performed to investigate the mechanism of sequence-specific recognition of the GGAA DNA core by the ETS domain. Employing the crystal structure of the Ets-1 ETS domain-DNA complex as a starting structure we carried out MD simulations of: (i). the complex between Ets-1 ETS domain and a 14 base-pair DNA containing GGAA core sequence (ETS-GGAA); (ii). the complex between the ETS domain and a DNA having single base-pair mutation, GGAG sequence (ETS-GGAG); and (iii). the 14 base-pair DNA alone (GGAA). Comparative analyses of the MD structures of ETS-GGAA and ETS-GGAG reveal that the DNA bending angles and the ETS domain-DNA phosphate interactions are similar in these complexes. These results support that the GGAA core sequence is distinguished from the mutated GGAG sequence by a direct readout mechanism in the Ets-1 ETS domain-DNA complex. Further analyses of the direct contacts in the interface between the helix-3 region of Ets-1 and the major groove of the core DNA sequence clearly show that the highly conserved arginine residues, Arg391 and Arg394, play a critical role in binding to the GGAA core sequence. These arginine residues make bidentate contacts with the nucleobases of GG dinucleotides in GGAA core sequence. In ETS-GGAA, the hydroxyl group of Tyr395 is hydrogen bonded to N7 nitrogen of A(3) (the third adenosine in the GGAA core), while the hydroxyl group makes a contact with N4 nitrogen of C(4') (the complementary nucleotide of the fourth guanosine G(4) in the GGAG sequence) in the ETS-GGAG complex. We have found that this difference in behavior of Tyr395 results in the relatively large motion of helix-3 in the ETS-GGAG complex, causing the collapse of bidentate contacts between Arg391/Arg394 and the GG dinucleotides in the GGAG sequence.