Osmolyte perturbation reveals conformational equilibria in spin‐labeled proteins

Recent evidence suggests that proteins at equilibrium can exist in a manifold of conformational substates, and that these substates play important roles in protein function. Therefore, there is great interest in identifying regions in proteins that are in conformational exchange. Electron paramagnetic resonance spectra of spin‐labeled proteins containing the nitroxide side chain (R1) often consist of two (or more) components that may arise from slow exchange between conformational substates (lifetimes > 100 ns). However, crystal structures of proteins containing R1 have shown that multicomponent spectra can also arise from equilibria between rotamers of the side chain itself. In this report, it is shown that these scenarios can be distinguished by the response of the system to solvent perturbation with stabilizing osmolytes such as sucrose. Thus, site‐directed spin labeling (SDSL) emerges as a new tool to explore slow conformational exchange in proteins of arbitrary size, including membrane proteins in a native‐like environment. Moreover, equilibrium between substates with even modest differences in conformation is revealed, and the simplicity of the method makes it suitable for facile screening of multiple proteins. Together with previously developed strategies for monitoring picosecond to millisecond backbone dynamics, the results presented here expand the timescale over which SDSL can be used to explore protein flexibility.     

Structural and mutational studies on an aldo‐keto reductase AKR5C3 from Gluconobacter oxydans

An aldo‐keto reductase AKR5C3 from Gluconobacter oxydans (designated as Gox0644) is a useful enzyme with various substrates, including aldehydes, diacetyl, keto esters, and α‐ketocarbonyl compounds. The crystal structures of AKR5C3 in apoform in complex with NADPH and the D53A mutant (AKR5C3^‐D53A) in complex with NADPH are presented herein. Structure comparison and site‐directed mutagenesis combined with biochemical kinetics analysis reveal that the conserved Asp53 in the AKR5C3 catalytic tetrad has a crucial role in securing active pocket conformation. The gain‐of‐function Asp53 to Ala mutation triggers conformational changes on the Trp30 and Trp191 side chains, improving NADPH affinity to AKR5C3, which helps increase catalytic efficiency. The highly conserved Trp30 and Trp191 residues interact with the nicotinamide moiety of NADPH and help form the NADPH‐binding pocket. The AKR5C3^‐W30A and AKR5C3^‐W191Y mutants show decreased activities, confirming that both residues facilitate catalysis. Residue Trp191 is in the loop structure, and the AKR5C3^‐W191Y mutant does not react with benzaldehyde, which might also determine substrate recognition. Arg192, which is involved in the substrate binding, is another important residue. The introduction of R192G increases substrate‐binding affinity by improving hydrophobicity in the substrate‐binding pocket. These results not only supplement the AKRs superfamily with crystal structures but also provide useful information for understanding the catalytic properties of AKR5C3 and guiding further engineering of this enzyme.     

Computer modeling of nitroxide spin labels on proteins

Electron paramagnetic resonance using site‐directed spin labeling can be used as an approach for determination of protein structures that are difficult to solve by other methods. One important aspect of this approach is the measurement of interlabel distances using the double electron–electron resonance (DEER) method. Interpretation of experimental data could be facilitated by a computational approach to calculation of interlabel distances. We describe an algorithm, PRONOX, for rapid computation of interlabel distances based on calculation of spin label conformer distributions at any site of a protein. The program incorporates features of the label distribution established experimentally, including weighting of favorable conformers of the label. Distances calculated by PRONOX were compared with new DEER distances for amphiphysin and annexin B12 and with published data for FCHo2 (F‐BAR), endophilin, and α‐synuclein, a total of 44 interlabel distances. The program reproduced these distances accurately (r² = 0.94, slope = 0.98). For 9 of the 11 distances for amphiphysin, PRONOX reproduced the experimental data to within 2.5 Å. The speed and accuracy of PRONOX suggest that the algorithm can be used for fitting to DEER data for determination of protein tertiary structure. © 2011 Wiley Periodicals, Inc. Biopolymers 97: 35–44, 2012.     

Crystal structures of ligand‐bound octaprenyl pyrophosphate synthase from Escherichia coli reveal the catalytic and chain‐length determining mechanisms

Octaprenyl pyrophosphate synthase (OPPs) catalyzes consecutive condensation reactions of one allylic substrate farnesyl pyrophosphate (FPP) and five homoallylic substrate isopentenyl pyrophosphate (IPP) molecules to form a C₄₀ long‐chain product OPP, which serves as a side chain of ubiquinone and menaquinone. OPPs belongs to the trans‐prenyltransferase class of proteins. The structures of OPPs from Escherichia coli were solved in the apo‐form as well as in complexes with IPP and a FPP thio‐analog, FsPP, at resolutions of 2.2–2.6 Å, and revealed the detailed interactions between the ligands and enzyme. At the bottom of the active‐site tunnel, M123 and M135 act in concert to form a wall which determines the final chain length. These results represent the first ligand‐bound crystal structures of a long‐chain trans‐prenyltransferase and provide new information on the mechanisms of catalysis and product chain elongation. Proteins 2015; 83:37–45. © 2014 Wiley Periodicals, Inc.     

Osmolytes modulate conformational exchange in solvent‐exposed regions of membrane proteins

Site‐directed spin labeling (SDSL) was used to investigate local structure and conformational exchange in two bacterial outer‐membrane TonB‐dependent transporters, BtuB and FecA. Protecting osmolytes, such as polyethylene glycols (PEGs) are known to modulate a substrate‐dependent conformational equilibrium in the energy coupling motif (Ton box) of BtuB. Here, we demonstrate that a segment that is N‐terminal to the Ton box in BtuB, is in conformational exchange between ordered and disordered states with or without substrate. Protecting osmolytes shift this equilibrium to favor the more ordered, folded state. However, a segment of BtuB that is C‐terminal to the Ton box that is not solvent exposed is insensitive to PEGs. Protecting osmolytes also modulate a conformational equilibrium in the Ton box of FecA, with larger molecular weight PEGs producing the largest shifts in the conformational free energy. These data indicate that solvent‐exposed regions of these transporters undergo conformational exchange and that regions of these transporters that are involved in protein–protein interactions sample multiple conformational substates. The sensitivity to solute provides an explanation for differences seen between two high‐resolution structures of BtuB, which each likely represent one conformation from a subset of states that are normally sampled by the protein. This work also illustrates how SDSL and osmolytes may be used to characterize and quantitate conformational equilibria in membrane proteins.     

Structures of yeast glutathione‐S‐transferase Gtt2 reveal a new catalytic type of GST family

Glutathione‐S‐transferases (GSTs) are ubiquitous detoxification enzymes that catalyse the conjugation of electrophilic substrates to glutathione. Here, we present the crystal structures of Gtt2, a GST of Saccharomyces cerevisiae, in apo and two ligand‐bound forms, at 2.23 Å, 2.20 Å and 2.10 Å, respectively. Although Gtt2 has the overall structure of a GST, the absence of the classic catalytic essential residues—tyrosine, serine and cysteine—distinguishes it from all other cytosolic GSTs of known structure. Site‐directed mutagenesis in combination with activity assays showed that instead of the classic catalytic residues, a water molecule stabilized by Ser129 and His123 acts as the deprotonator of the glutathione sulphur atom. Furthermore, only glycine and alanine are allowed at the amino‐terminus of helix‐α1 because of stereo‐hindrance. Taken together, these results show that yeast Gtt2 is a novel atypical type of cytosolic GST.     

Structure and activity of the NAD(P)+‐dependent succinate semialdehyde dehydrogenase YneI from Salmonella typhimurium

Aldehyde dehydrogenases are found in all organisms and play an important role in the metabolic conversion and detoxification of endogenous and exogenous aldehydes. Genomes of many organisms including Escherichia coli and Salmonella typhimurium encode two succinate semialdehyde dehydrogenases with low sequence similarity and different cofactor preference (YneI and GabD). Here, we present the crystal structure and biochemical characterization of the NAD(P)⁺‐dependent succinate semialdehyde dehydrogenase YneI from S. typhimurium. This enzyme shows high activity and affinity toward succinate semialdehyde and exhibits substrate inhibition at concentrations of SSA higher than 0.1 mM. YneI can use both NAD⁺ and NADP⁺ as cofactors, although affinity to NAD⁺ is 10 times higher. High resolution crystal structures of YneI were solved in a free state (1.85 Å) and in complex with NAD⁺ (1.90 Å) revealing a two domain protein with the active site located in the interdomain interface. The NAD⁺ molecule is bound in the long channel with its nicotinamide ring positioned close to the side chain of the catalytic Cys268. Site‐directed mutagenesis demonstrated that this residue, as well as the conserved Trp136, Glu365, and Asp426 are important for activity of YneI, and that the conserved Lys160 contributes to the enzyme preference to NAD⁺. Our work has provided further insight into the molecular mechanisms of substrate selectivity and activity of succinate semialdehyde dehydrogenases. © 2012 Wiley Periodicals, Inc.     

Structure‐based design of novel Chlamydomonas reinhardtii D1‐D2 photosynthetic proteins for herbicide monitoring

The D1‐D2 heterodimer in the reaction center core of phototrophs binds the redox plastoquinone cofactors, Q_A and Q_B, the terminal acceptors of the photosynthetic electron transfer chain in the photosystem II (PSII). This complex is the target of the herbicide atrazine, an environmental pollutant competitive inhibitor of Q_B binding, and consequently it represents an excellent biomediator to develop biosensors for pollutant monitoring in ecosystems. In this context, we have undertaken a study of the Chlamydomonas reinhardtii D1‐D2 proteins aimed at designing site directed mutants with increased affinity for atrazine. The three‐dimensional structure of the D1 and D2 proteins from C. reinhardtii has been homology modeled using the crystal structure of the highly homologous Thermosynechococcus elongatus proteins as templates. Mutants of D1 and D2 were then generated in silico and the atrazine binding affinity of the mutant proteins has been calculated to predict mutations able to increase PSII affinity for atrazine. The computational approach has been validated through comparison with available experimental data and production and characterization of one of the predicted mutants. The latter analyses indicated an increase of one order of magnitude of the mutant sensitivity and affinity for atrazine as compared to the control strain. Finally, D1‐D2 heterodimer mutants were designed and selected which, according to our model, increase atrazine binding affinity by up to 20 kcal/mol, representing useful starting points for the development of high affinity biosensors for atrazine.     

Crystallographic dissection of the thermal motion of protein-sugar complex

The crystal structure of turkey egg lysozyme (TEL) complexed with di-N-acetylchitobiose (NAG2) was refined at 1.19 A resolution by the full-matrix least-squares method with anisotropic temperature factors, and its thermal motion was evaluated by the TLS method. The average ESDs of atomic parameters of nonhydrogen atoms were 0.030 A for coordinates and 0.025 A(2) for anisotropic temperature factors. The active site cleft of TEL binds the alpha-anomer of NAG2 in a nonproductive binding mode with its pyranose rings parallel to a beta-sheet. The TEL structure was compared with the re-refined 1.12 A structure of native TEL. The RMS difference for equivalent Calpha atoms was 0.103 A and a relatively large difference was observed in the region of residues 104-125 rather than in the beta-sheet region where NAG2 was bound. In contrast, the temperature factor of the beta-sheet region was significantly decreased by the NAG2 binding. The TLS model that describes the rigid body motion in translation, libration, and screw motion was adopted for the evaluation of the molecular motion of TEL and NAG2, and the TLS parameters were determined by the least-squares fit to U(ij). The contribution of the external motion of TEL was estimated to be 55.8% of the observed temperature factor for the native structure and 45.9% for the NAG2 complex. The internal motion of TEL represented with atomic thermal ellipsoids was very similar between the native and complex structures except the NAG2 binding region. In the structure of NAG2, the rigid body motion dominates the thermal motion. The center of rotation of NAG2, 4.45A far from the center of gravity, is on the nitrogen atom of the acetylamino group that is hydrogen bonded to the main-chain peptide groups of Asn49 and Ala107. The rigid body motion of NAG2 indicates that the acetylamino group is most strongly bound to the active site, and the recognition of this group is a crucial step of the substrate binding.     

Quantitative evaluation of His‐tag purification and immunoprecipitation of tristetraprolin and its mutant proteins from transfected human cells

Histidine (His)‐tag is widely used for affinity purification of recombinant proteins, but the yield and purity of expressed proteins are quite different. Little information is available about quantitative evaluation of this procedure. The objective of this study was to evaluate His‐tag procedure quantitatively and to compare it with immunoprecipitation using radiolabeled tristetraprolin (TTP), a zinc finger protein with anti‐inflammatory property. Human embryonic kidney 293 cells were transfected with wild‐type and nine mutant plasmids with single or multiple phosphorylation site mutation(s) in His‐TTP. These proteins were expressed and mainly localized in the cytosol of transfected cells by immunocytochemistry and confocal microscopy. His‐TTP proteins were purified by Ni‐NTA beads with imidazole elution or precipitated by TTP antibodies from transfected cells after being labeled with [³²P]‐orthophosphate. The results showed that (1) His‐tag purification was more effective than immunoprecipitation for TTP purification; (2) mutations in TTP increased the yield of His‐TTP by both purification procedures; and (3) mutations in TTP increased the binding affinity of mutant proteins for Ni‐NTA beads. These findings suggest that bioengineering phosphorylation sites in proteins can increase the production of recombinant proteins. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Hexafluoroisopropanol induces self‐assembly of β‐amyloid peptides into highly ordered nanostructures

Deposition of insoluble fibrillar aggregates of β‐amyloid (Aβ) peptides in the brain is a hallmark of Alzheimer's disease. Apart from forming fibrils, these peptides also exist as soluble aggregates. Fibrillar and a variety of nonfibrillar aggregates of Aβ have also been obtained in vitro. Hexafluoroisopropanol (HFIP) has been widely used to dissolve Aβ and other amyloidogenic peptides. In this study, we show that the dissolution of Aβ40, 42, and 43 in HFIP followed by drying results in highly ordered aggregates. Although α‐helical conformation is observed, it is not stable for prolonged periods. Drying after prolonged incubation of Aβ40, 42, and 43 peptides in HFIP leads to structural transition from α‐helical to β‐conformation. The peptides form short fibrous aggregates that further assemble giving rise to highly ordered ring‐like structures. Aβ16–22, a highly amyloidogenic peptide stretch from Aβ, also formed very similar rings when dissolved in HFIP and dried. HFIP could not induce α‐helical conformation in Aβ16–22, and rings were obtained from freshly dissolved peptide. The rings formed by Aβ40, 42, 43, and Aβ16–22 are composed of the peptides in β‐conformation and cause enhancement in thioflavin T fluorescence, suggesting that the molecular architecture of these structures is amyloid‐like. Our results clearly indicate that dissolution of Aβ40, 42 and 43 and the amyloidogenic fragment Aβ16–22 in HFIP results in the formation of annular amyloid‐like structures. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Improving GPX activity of selenium‐containing human single‐chain Fv antibody by site‐directed mutation based on the structural analysis

Glutathione peroxidase (GPX) is one of the important members of the antioxidant enzyme family. It can catalyze the reduction of hydroperoxides with glutathione to protect cells against oxidative damage. In previous studies, we have prepared the human catalytic antibody Se‐scFv‐B3 (selenium‐containing single‐chain Fv fragment of clone B3) with GPX activity by incorporating a catalytic group Sec (selenocysteine) into the binding site using chemical mutation; however, its activity was not very satisfying. In order to try to improve its GPX activity, structural analysis of the scFv‐B3 was carried out. A three‐dimensional (3D) structure of scFv‐B3 was constructed by means of homology modeling and binding site analysis was carried out. Computer‐aided docking and energy minimization (EM) calculations of the antibody‐GSH (glutathione) complex were also performed. From these simulations, Ala44 and Ala180 in the candidate binding sites were chosen to be mutated to serines respectively, which can be subsequently converted into the catalytic Sec group. The two mutated protein and wild type of the scFv were all expressed in soluble form in Escherichia coli Rosetta and purified by Ni²⁺‐immobilized metal affinity chromatography (IMAC), then transformed to selenium‐containing catalytic antibody with GPX activity by chemical modification of the reactive serine residues. The GPX activity of the mutated catalytic antibody Se‐scFv‐B3‐A180S was significantly increased compared to the original Se‐scFv‐B3. Copyright © 2009 John Wiley & Sons, Ltd.     

Structural determinants of nitroxide motion in spin-labeled proteins: solvent-exposed sites in helix B of T4 lysozyme

Site-directed spin labeling provides a means for exploring structure and dynamics in proteins. To interpret the complex EPR spectra that often arise, it is necessary to characterize the rotamers of the spin-labeled side chain and the interactions they make with the local environment in proteins of known structure. For this purpose, crystal structures have been determined for T4 lysozyme bearing a nitroxide side chain (R1) at the solvent-exposed helical sites 41 and 44 in the B helix. These sites are of particular interest in that the corresponding EPR spectra reveal two dynamic states of R1, one of which is relatively immobilized suggesting interactions of the nitroxide with the environment. The crystal structures together with the effect of mutagenesis of nearest neighbors on the motion of R1 suggest intrahelical interactions of 41R1 with the i + 4 residue and of 44R1 with the i + 1 residue. Such interactions appear to be specific to particular rotamers of the R1 side chain.     

Structural and catalytic role of two conserved tyrosines in Delta-class glutathione S-transferase from Locusta migratoria

Glutathione S-transferases (GSTs) are an important family of detoxifying enzymes and play a key role in pesticide resistance in the insect. Tyrosine is essential for its detoxification function. In the present study, two conserved tyrosine residues are located at positions 108 and 116 in H-site of LmGSTD1. To elucidate how the two residues participate in the catalytic process and keeping structural stability, four mutants, Y108A, Y108E, Y116A, and Y116E, were generated. It was found that the four mutants affected the specific activity of LmGSTD1 in various degrees, depending on the types of substrate and reaction mechanism. Steady-state kinetics assay revealed that Y108E and Y116E had a significant influence on GSH-binding ability, which indicates the two tyrosine residues of H-site contribute to topology rearrangement of G-site. Both Y116A and Y116E exhibited lower CDNB-binding affinity, suggesting that Y116 takes part in hydrophobic substrate binding. The thermostability assay, intrinsic, and 8-anilino-1-naphthalenesulfonic acid (ANS) florescence results showed that the two tyrosine residues were involved in regulation of active-site conformation. Finally, homology modeling provided evidence that the two tyrosines in H-site participate in hydrophobic substrate binding. Furthermore, Y108 is closer to the S atom of S-hexylglutathione. In conclusion, the two tyrosines in LmGSTD1 are important residues in both the catalytic process and protein stability.     

Hot‐spot analysis to dissect the functional protein–protein interface of a tRNA‐modifying enzyme

Interference with protein–protein interactions of interfaces larger than 1500 Ų by small drug‐like molecules is notoriously difficult, particularly if targeting homodimers. The tRNA modifying enzyme Tgt is only functionally active as a homodimer. Thus, blocking Tgt dimerization is a promising strategy for drug therapy as this protein is key to the development of Shigellosis. Our goal was to identify hot‐spot residues which, upon mutation, result in a predominantly monomeric state of Tgt. The detailed understanding of the spatial location and stability contribution of the individual interaction hot‐spot residues and the plasticity of motifs involved in the interface formation is a crucial prerequisite for the rational identification of drug‐like inhibitors addressing the respective dimerization interface. Using computational analyses, we identified hot‐spot residues that contribute particularly to dimer stability: a cluster of hydrophobic and aromatic residues as well as several salt bridges. This in silico prediction led to the identification of a promising double mutant, which was validated experimentally. Native nano‐ESI mass spectrometry showed that the dimerization of the suggested mutant is largely prevented resulting in a predominantly monomeric state. Crystal structure analysis and enzyme kinetics of the mutant variant further support the evidence for enhanced monomerization and provide first insights into the structural consequences of the dimer destabilization. Proteins 2014; 82:2713–2732. © 2014 Wiley Periodicals, Inc.     

Prediction of protein–protein interaction sites from weakly homologous template structures using meta‐threading and machine learning

The identification of protein–protein interactions is vital for understanding protein function, elucidating interaction mechanisms, and for practical applications in drug discovery. With the exponentially growing protein sequence data, fully automated computational methods that predict interactions between proteins are becoming essential components of system‐level function inference. A thorough analysis of protein complex structures demonstrated that binding site locations as well as the interfacial geometry are highly conserved across evolutionarily related proteins. Because the conformational space of protein–protein interactions is highly covered by experimental structures, sensitive protein threading techniques can be used to identify suitable templates for the accurate prediction of interfacial residues. Toward this goal, we developed eFindSite^PPI, an algorithm that uses the three‐dimensional structure of a target protein, evolutionarily remotely related templates and machine learning techniques to predict binding residues. Using crystal structures, the average sensitivity (specificity) of eFindSite^PPI in interfacial residue prediction is 0.46 (0.92). For weakly homologous protein models, these values only slightly decrease to 0.40–0.43 (0.91–0.92) demonstrating that eFindSite^PPI performs well not only using experimental data but also tolerates structural imperfections in computer‐generated structures. In addition, eFindSite^PPI detects specific molecular interactions at the interface; for instance, it correctly predicts approximately one half of hydrogen bonds and aromatic interactions, as well as one third of salt bridges and hydrophobic contacts. Comparative benchmarks against several dimer datasets show that eFindSite^PPI outperforms other methods for protein‐binding residue prediction. It also features a carefully tuned confidence estimation system, which is particularly useful in large‐scale applications using raw genomic data. eFindSite^PPI is freely available to the academic community at http://www.brylinski.org/efindsiteppi . Copyright © 2014 John Wiley & Sons, Ltd.     

The importance of valine 114 in ligand binding in β2‐adrenergic receptor

G‐protein coupled receptors (GPCRs) are transmembrane signaling molecules, with a majority of them performing important physiological roles. β₂‐Adrenergic receptor (β₂‐AR) is a well‐studied GPCRs that mediates natural responses to the hormones adrenaline and noradrenaline. Analysis of the ligand‐binding region of β₂‐AR using the recently solved high‐resolution crystal structures revealed a number of highly conserved amino acids that might be involved in ligand binding. However, detailed structure‐function studies on some of these residues have not been performed, and their role in ligand binding remains to be elucidated. In this study, we have investigated the structural and functional role of a highly conserved residue valine 114, in hamster β₂‐AR by site‐directed mutagenesis. We replaced V114 in hamster β₂‐AR with a number of amino acid residues carrying different functional groups. In addition to the complementary substitutions V114I and V114L, the V114C and V114E mutants also showed significant ligand binding and agonist dependent G‐protein activation. However, the V114G, V114T, V114S, and V114W mutants failed to bind ligand in a specific manner. Molecular modeling studies were conducted to interpret these results in structural terms. We propose that the replacement of V114 influences not only the interaction of the ethanolamine side‐chains but also the aryl‐ring of the ligands tested. Results from this study show that the size and orientation of the hydrophobic residue at position V114 in β₂‐AR affect binding of both agonists and antagonists, but it does not influence the receptor expression or folding.     

Structural basis for the observed differential magnetic anisotropic tensorial values in calcium binding proteins

Lanthanide ions (Ln(3+)), which have ionic radii similar to those of Ca(2+), can displace the latter in a calcium binding protein, without affecting its tertiary structure. The paramagnetic Ln(3+) possesses large anisotropic magnetic susceptibilities and produce pseudocontact shifts (PCSs), which have r(-3) dependence. The PCS can be seen for spins as far as 45 A from the paramagnetic ion. They aid in structure refinement of proteins by providing long-range distance constraints. Besides, they can be used to determine the interdomain orientation in multidomain proteins. This is particularly important in the context of a calcium binding protein from Entamoeba histolytica (EhCaBP), which consists of two globular domains connected by a flexible linker region containing 8 residues. As a first step to obtain the interdomain orientation in EhCaBP, a suite of 2D and 3D heteronuclear experiments were recorded on EhCaBP by displacing calcium with Ce(3+), Ho(3+), Er(3+), Tm(3+), Dy(3+), and Yb(3+) ions in separate experiments, and the PCS of (1)H(N) and (15)N spins were measured. Such data have been used in the refinement of the individual domain structures of the protein in parallel with the calculation of the respective magnetic anisotropy tensorial values, which differ substantially (2.1-2.8 times) from what is found in other Ca(2+) binding loops. This study provides a structural basis for such variations in the magnetic anisotropy tensorial values.     

Rigid‐body motions of interacting proteins dominate multispecific binding of ubiquitin in a shape‐dependent manner

To understand the dynamic aspects of multispecificity of ubiquitin, we studied nine ubiquitin–ligand (partner protein) complexes by normal mode analysis based on an elastic network model. The coupling between ubiquitin and ligand motions was analyzed by decomposing it into rigid‐body (external) and vibrational (internal) motions of each subunit. We observed that in total the external motions in one of the subunits largely dominated the coupling. The combination of external motions of ubiquitin and the ligands showed general trends of rotations and translations. Moreover, we observed that the rotational motions of ubiquitin were correlated to the ligand orientations. We also identified ubiquitin atomic vibrations that differentiated the orientation of the ligand molecule. We observed that the extents of coupling were correlated to the shapes of the ligands, and this trend was more pronounced when the coupling involved vibrational motions of the ligand. In conclusion, an intricate interplay between internal and external motions of ubiquitin and the ligands help understand the dynamics of multispecificity, which is mostly guided by the shapes of the ligands and the complex. Proteins 2014; 82:77–89. © 2013 Wiley Periodicals, Inc.