Domain Interface Research Articles

Prediction of New Stabilizing Mutations Based on Mechanistic Insights from Markov State Models

Protein stabilization is fundamental to enzyme function and evolution, yet understanding the determinants of a protein's stability remains a challenge. This is largely due to a shortage of atomically-detailed models for the ensemble of relevant protein conformations and their relative populations. For example, the M182T substitution in TEM β-lactamase, an enzyme that confers antibiotic resistance to bacteria, is stabilizing but the precise mechanism remains unclear. Here, we employ Markov state models (MSMs) to uncover how M182T shifts the distribution of different structures that TEM adopts. We find that M182T stabilizes a helix that is a key component of a domain interface. We then predict the effects of other mutations, including a novel stabilizing mutation, and experimentally test our predictions using a combination of stability measurements, crystallography, NMR, and in vivo measurements of bacterial fitness. We expect our insights and methodology to provide a valuable foundation for protein design.

Highly Asymmetric Phase Behaviors of Polyhedral Oligomeric Silsesquioxane-Based Multiheaded Giant Surfactants.

This work reports the molecular design, synthesis, and systematic study on the bulk self-assembly behaviors of three series of polyhedral oligomeric silsesquioxane (POSS)-based multiheaded giant surfactants XDPOSS-PSn (X = 2, 3, and 4), which are composed of two, three, or four hydrophilic hydroxyl-group-functionalized DPOSS cages attached via one junction point to a hydrophobic polystyrene (PS) chain. These series of hybrid polymeric amphiphiles with precisely defined chemical structure and controllable molecular architecture are synthesized by the sequential usage of "click" reactions. By tuning molecular weights of the PS tail, we established full phase diagrams of XDPOSS-PSn as a function of the volume fractions of PS chains (VfPS). We found that the self-assembled structures were greatly influenced by the molecular architecture. Strikingly, our results showed that the lamellar morphology, which usually existed at relatively symmetric compositions in common diblock copolymers, became the thermodynamically stable phase in the 3DPOSS-PSn and 4DPOSS-PSn samples even at an asymmetric composition up to VfPS = 0.842, with the ratio between the thicknesses of PS and DPOSS lamellae up to 5.32. This unusual phenomenon induced by molecular architectural variation could be explained by the large cross-sectional area of DPOSS cages at the nanophase-separated domain interface and high elastic deformation energy of clustered DPOSS cages which have relatively rigid conformation. The unique bulk self-assembly behaviors in our POSS-based multiheaded giant surfactants provide insights in developing hybrid nanomaterials toward unconventional nanostructures.

SUMOylation Regulates Transcription by the Progesterone Receptor A Isoform in a Target Gene Selective Manner.

Luminal breast cancers express estrogen (ER) and progesterone (PR) receptors, and respond to endocrine therapies. However, some ER+PR+ tumors display intrinsic or acquired resistance, possibly related to PR. Two PR isoforms, PR-A and PR-B, regulate distinct gene subsets that may differentially influence tumor fate. A high PR-A:PR-B ratio is associated with poor prognosis and tamoxifen resistance. We speculate that excessive PR-A marks tumors that will relapse early. Here we address mechanisms by which PR-A regulate transcription, focusing on SUMOylation. We use receptor mutants and synthetic promoter/reporters to show that SUMOylation deficiency or the deSUMOylase SENP1 enhance transcription by PR-A, independent of the receptors’ dimerization interface or DNA binding domain. De-SUMOylation exposes the agonist properties of the antiprogestin RU486. Thus, on synthetic promoters, SUMOylation functions as an independent brake on transcription by PR-A. What about PR-A SUMOylation of endogenous human breast cancer genes? To study these, we used gene expression profiling. Surprisingly, PR-A SUMOylation influences progestin target genes differentially, with some upregulated, others down-regulated, and others unaffected. Hormone-independent gene regulation is also PR-A SUMOylation dependent. Several SUMOylated genes were analyzed in clinical breast cancer database. In sum, we show that SUMOylation does not simply repress PR-A. Rather it regulates PR-A activity in a target selective manner including genes associated with poor prognosis, shortened survival, and metastasis.

Stochastic Domain Decomposition via Moment Minimization

Propagating uncertainty accurately across different domains in multiscale physical systems with vastly different correlation lengths is of fundamental importance in stochastic simulations. We propose a new method to address this issue, namely, the stochastic domain decomposition via moment minimization (SDD-MM). Specifically, we develop a new moment minimizing interface condition to match the stochastic solutions at the interface of the nonoverlapping domains. Unlike other stochastic domain decomposition methods, the proposed method serves as a general framework that works with heterogeneous local stochastic solvers and does not rely on accessing global random trajectories, which are typically not available in realistic multiscale simulations. We analyze the computational complexity of the method and we quantify the contributing errors. The convergence property of SDD-MM is tested in several examples that include the stochastic reaction equation, Fisher's equation, as well as a two-dimensional Allen--Cahn equation. We observe good performance of the method for nonlinear problems as well as problems with different correlation lengths.

Resistance to pyridine-based inhibitor KF116 reveals an unexpected role of integrase in HIV-1 Gag-Pol polyprotein proteolytic processing

The pyridine-based multimerization selective HIV-1 integrase (IN) inhibitors (MINIs) are a distinct subclass of allosteric IN inhibitors. MINIs potently inhibit HIV-1 replication during virion maturation by inducing hyper- or aberrant IN multimerization but are largely ineffective during the early steps of viral replication. Here, we investigated the mechanism for the evolution of a triple IN substitution (T124N/V165I/T174I) that emerges in cell culture with a representative MINI, KF116. We show that HIV-1 NL4-3(IN T124N/V165I/T174I) confers marked (>2000-fold) resistance to KF116. Two IN substitutions (T124N/T174I) directly weaken inhibitor binding at the dimer interface of the catalytic core domain but at the same time markedly impair HIV-1 replication capacity. Unexpectedly, T124N/T174I IN substitutions inhibited proteolytic processing of HIV-1 polyproteins Gag and Gag-Pol, resulting in immature virions. Strikingly, the addition of the third IN substitution (V165I) restored polyprotein processing, virus particle maturation, and significant levels of replication capacity. These results reveal an unanticipated role of IN for polyprotein proteolytic processing during virion morphogenesis. The complex evolutionary pathway for the emergence of resistant viruses, which includes the need for the compensatory V165I IN substitution, highlights a relatively high genetic barrier exerted by MINI KF116. Additionally, we have solved the X-ray structure of the drug-resistant catalytic core domain protein, which provides means for rational development of second-generation MINIs.

Coordination and redox state–dependent structural changes of the heme-based oxygen sensor AfGcHK associated with intraprotein signal transduction

The heme-based oxygen sensor histidine kinase AfGcHK is part of a two-component signal transduction system in bacteria. O2 binding to the Fe(II) heme complex of its N-terminal globin domain strongly stimulates autophosphorylation at His183 in its C-terminal kinase domain. The 6-coordinate heme Fe(III)-OH- and -CN- complexes of AfGcHK are also active, but the 5-coordinate heme Fe(II) complex and the heme-free apo-form are inactive. Here, we determined the crystal structures of the isolated dimeric globin domains of the active Fe(III)-CN- and inactive 5-coordinate Fe(II) forms, revealing striking structural differences on the heme-proximal side of the globin domain. Using hydrogen/deuterium exchange coupled with mass spectrometry to characterize the conformations of the active and inactive forms of full-length AfGcHK in solution, we investigated the intramolecular signal transduction mechanisms. Major differences between the active and inactive forms were observed on the heme-proximal side (helix H5), at the dimerization interface (helices H6 and H7 and loop L7) of the globin domain and in the ATP-binding site (helices H9 and H11) of the kinase domain. Moreover, separation of the sensor and kinase domains, which deactivates catalysis, increased the solvent exposure of the globin domain-dimerization interface (helix H6) as well as the flexibility and solvent exposure of helix H11. Together, these results suggest that structural changes at the heme-proximal side, the globin domain-dimerization interface, and the ATP-binding site are important in the signal transduction mechanism of AfGcHK. We conclude that AfGcHK functions as an ensemble of molecules sampling at least two conformational states.

Structural and Functional Characterization of a Hole-Hole Homodimer Variant in a "Knob-Into-Hole" Bispecific Antibody.

Bispecific antibodies have great potential to be the next-generation biotherapeutics due to their ability to simultaneously recognize two different targets. Compared to conventional monoclonal antibodies, knob-into-hole bispecific antibodies face unique challenges in production and characterization due to the increase in variant possibilities, such as homodimerization in covalent and noncovalent forms. In this study, a storage- and pH-sensitive hydrophobic interaction chromatography (HIC) profile change was observed for the hole-hole homodimer, and the multiple HIC peaks were explored and shown to be conformational isomers. We combined traditional analytical methods with hydrogen/deuterium exchange mass spectrometry (HDX MS), native mass spectrometry, and negative-staining electron microscopy to comprehensively characterize the hole-hole homodimer. HDX MS revealed conformational changes at the resolution of a few amino acids overlapping the CH2-CH3 domain interface. Conformational heterogeneity was also assessed by HDX MS isotopic distribution. The hole-hole homodimer was demonstrated to adopt a more homogeneous conformational distribution during storage. This conformational change is likely caused by a lack of CH3 domain dimerization (due to the three "hole" point mutations), resulting in a unique storage- and pH-dependent conformational destabilization and refolding of the hole-hole homodimer Fc. Compared with the hole-hole homodimer under different storage conditions, the bispecific heterodimer, guided by the knob-into-hole assembly, proved to be a stable conformation with homogeneous distribution, confirming its high quality as a desired therapeutic. Functional studies by antigen binding and neonatal Fc receptor (FcRn) binding correlated very well with the structural characterization. Comprehensive interpretation of the results has provided a better understanding of both the homodimer variant and the bispecific molecule.

Toward a Novel Drug To Target the EGF-EGFR Interaction: Design of Metabolically Stable Bicyclic Peptides.

In cancer, proliferation of malignant cells is driven by overactivation of growth-signalling mechanisms, such as the epidermal growth factor receptor (EGFR) pathway. Despite its therapeutic relevance, the EGF-EGFR interaction has remained elusive to inhibition by synthetic molecules, mostly as a result of its large size and lack of binding pockets and cavities. Designed peptides, featuring cyclic motifs and other structural constraints, have the potential to modulate such challenging protein-protein interactions (PPIs). Herein, we present the structure-based design of a series of bicyclic constrained peptides that mimic an interface domain of EGFR and inhibit the EGF-EGFR interaction by targeting the smaller partner (i.e., EGF). This design process was guided by the integrated use of in silico methods and biophysical techniques, such as NMR spectroscopy and surface acoustic wave. The best analogues were able to reduce selectively the viability of EGFR+ human cancer cells. In addition to their efficacy, these bicyclic peptides are endowed with exceptional stability and metabolic resistance-two features that make them suitable candidates for in vivo applications.

Design and verification of halogen-bonding system at the complex interface of human fertilization-related MUP PDZ5 domain with CAMK’s C-terminal peptide

Calmodulin-dependent protein kinase (CAMK) is physiologically activated in fertilized human oocytes and is involved in the Ca2+ response pathways that link the fertilization calmodulin signal to meiosis resumption and cortical granule exocytosis. The kinase has an unstructured C-terminal tail that can be recognized and bound by the PDZ5 domain of its cognate partner, the multi-PDZ domain protein (MUP). In the current study, we reported a rational biomolecular design of halogen-bonding system at the complex interface of CAMK’s C-terminal peptide with MUP PDZ5 domain by using high-level computational approaches. Four organic halogens were employed as atom probes to explore the structural geometry and energetic property of designed halogen bonds in the PDZ5–peptide complex. It was found that the heavier halogen elements such as bromine Br and iodine I can confer stronger halogen bond but would cause bad atomic contacts and overlaps at the complex interface, while fluorine F cannot form effective halogen bond in the complex. In addition, the halogen substitution at different positions of peptide’s aromatic ring would result in distinct effects on the halogen-bonding system. The computational findings were then verified by using fluorescence analysis; it is indicated that the halogen type and substitution position play critical role in the interaction strength of halogen bonds, and thus the PDZ5–peptide binding affinity can be improved considerably by optimizing their combination.

Prediction of New Stabilizing Mutations Based on Mechanistic Insights from Markov State Models.

Protein stabilization is fundamental to enzyme function and evolution, yet understanding the determinants of a protein’s stability remains a challenge. This is largely due to a shortage of atomically detailed models for the ensemble of relevant protein conformations and their relative populations. For example, the M182T substitution in TEM β-lactamase, an enzyme that confers antibiotic resistance to bacteria, is stabilizing but the precise mechanism remains unclear. Here, we employ Markov state models (MSMs) to uncover how M182T shifts the distribution of different structures that TEM adopts. We find that M182T stabilizes a helix that is a key component of a domain interface. We then predict the effects of other mutations, including a novel stabilizing mutation, and experimentally test our predictions using a combination of stability measurements, crystallography, NMR, and in vivo measurements of bacterial fitness. We expect our insights and methodology to provide a valuable foundation for protein design.

Identification of Novel Allosteric Modulators of Glutamate Transporter EAAT2.

Dysfunction of excitatory amino acid transporters (EAATs) has been implicated in the pathogenesis of various neurological disorders, such as stroke, brain trauma, epilepsy, and neurodegenerative diseases, among others. EAAT2 is the main subtype responsible for glutamate clearance in the brain, having a key role in regulating transmission and preventing excitotoxicity. Therefore, compounds that increase the expression or activity of EAAT2 have therapeutic potential for neuroprotection. Previous studies identified molecular determinants for EAAT2 transport stimulation in a structural domain that lies at the interface of the rigid trimerization domain and the central substrate binding transport domain. In this work, a hybrid structure based approach was applied, based on this molecular domain, to create a high-resolution pharmacophore. Subsequently, virtual screening of a library of small molecules was performed, identifying 10 hit molecules that interact at the proposed domain. Among these, three compounds were determined to be activators, four were inhibitors, and three had no effect on EAAT2-mediated transport in vitro. Further characterization of the two best ranking EAAT2 activators for efficacy, potency, and selectivity for glutamate over monoamine transporters subtypes and NMDA receptors and for efficacy in cultured astrocytes is demonstrated. Mutagenesis studies suggest that the EAAT2 activators interact with residues forming the interface between the trimerization and transport domains. These compounds enhance the glutamate translocation rate, with no effect on substrate interaction, suggesting an allosteric mechanism. The identification of these novel positive allosteric modulators of EAAT2 offers an innovative approach for the development of therapies based on glutamate transport enhancement.

Structural basis for arginine methylation-independent recognition of PIWIL1 by TDRD2

The P-element-induced wimpy testis (PIWI)-interacting RNA (piRNA) pathway plays a central role in transposon silencing and genome protection in the animal germline. A family of Tudor domain proteins regulates the piRNA pathway through direct Tudor domain-PIWI interactions. Tudor domains are known to fulfill this function by binding to methylated PIWI proteins in an arginine methylation-dependent manner. Here, we report a mechanism of methylation-independent Tudor domain-PIWI interaction. Unlike most other Tudor domains, the extended Tudor domain of mammalian Tudor domain-containing protein 2 (TDRD2) preferentially recognizes an unmethylated arginine-rich sequence from PIWI-like protein 1 (PIWIL1). Structural studies reveal an unexpected Tudor domain-binding mode for the PIWIL1 sequence in which the interface of Tudor and staphylococcal nuclease domains is primarily responsible for PIWIL1 peptide recognition. Mutations disrupting the TDRD2-PIWIL1 interaction compromise piRNA maturation via 3'-end trimming in vitro. Our work presented here reveals the molecular divergence of the interactions between different Tudor domain proteins and PIWI proteins.

Un-gating and allosteric modulation of a pentameric ligand-gated ion channel captured by molecular dynamics.

Pentameric ligand-gated ion channels (pLGICs) mediate intercellular communication at synapses through the opening of an ion pore in response to the binding of a neurotransmitter. Despite the increasing availability of high-resolution structures of pLGICs, a detailed understanding of the functional isomerization from closed to open (gating) and back is currently missing. Here, we provide the first atomistic description of the transition from open to closed (un-gating) in the glutamate-gated chloride channel (GluCl) from Caenorhabditis Elegans. Starting with the active-state structure solved in complex with the neurotransmitter L-glutamate and the positive allosteric modulator (PAM) ivermectin, we analyze the spontaneous relaxation of the channel upon removal of ivermectin by explicit solvent/membrane Molecular Dynamics (MD) simulations. The μs-long trajectories support the conclusion that ion-channel deactivation is mediated by two distinct quaternary transitions, i.e. a global receptor twisting followed by the radial expansion (or blooming) of the extracellular domain. At variance with previous models, we show that pore closing is exclusively regulated by the global twisting, which controls the position of the β1-β2 loop relative to the M2-M3 loop at the EC/TM domain interface. Additional simulations with L-glutamate restrained to the crystallographic binding mode and ivermectin removed indicate that the same twisting isomerization is regulated by agonist binding at the orthosteric site. These results provide a structural model for gating in pLGICs and suggest a plausible mechanism for the pharmacological action of PAMs in this neurotransmitter receptor family. The simulated un-gating converges to the X-ray structure of GluCl resting state both globally and locally, demonstrating the predictive character of state-of-art MD simulations.

Molecular modeling on HIF-2α–ARNT dimer destabilization caused by R171A and/or V192D mutations in HIF-2α

Oxygen homeostasis in normal and tumor cells is mediated by hypoxia-inducible factors (HIFs), which are active as heterodimer complexes, such as HIF-2α–aryl hydrocarbon receptor nuclear translocator (ARNT) and HIF-1α–ARNT. A series of mutations on the interfaces between HIF-2α and ARNT and on the domain–domain interface within HIF-2α has been reported to exert varying effects on HIF-2α–ARNT dimerization. In the present study, molecular dynamic simulations were conducted to evaluate HIF-2α mutations, namely R171A, V192D, and R171A/V192D, which are not involved in the interaction with ARNT but impede HIF-2α–ARNT dimerization. Our results indicate that these mutations induct local conformation leading to a shortened (by V192D) or widened (by R171A and R171A/V192D) Y91–E346 separation distance, where E346 and Y91 are located on the HIF-2α and interact with ARNT according to electrostatic and geometrical shape complementarity, respectively.

A rational approach to enhancing antibody Fc homodimer formation for robust production of antibody mixture in a single cell line

Combinations of different antibodies have been shown to be more effective for managing certain diseases than monotherapy. Co-expression of the antibody mixture in a single cell line is key to reducing complexity during antibody development and manufacturing. However, co-transfection of multiple light and heavy chains into cells often leads to production of mismatched, heterodimeric by-products that are inactive, making the development of co-expression systems that robustly and efficiently produce highly active antibody mixtures a high priority. In this study, we modified the CH3 domain interface of the antibody fragment crystallizable (Fc) region by changing several charge pairs to create electrostatic interactions favoring Fc homodimer formation and disfavoring Fc heterodimer formation. When co-expressed, these modified antibodies with altered charge polarity across the Fc dimer interface preferentially formed homodimers that fully preserved the functions of each component, rather than inactive heterodimers whose formation was reduced because of rationally designed repulsive interactions. We designed eight different combinations and experimentally screened the best one, which enabled us to produce a binary antibody mixture against the EGF receptor with a minimal heterodimer contaminant. We further determined the crystal structure of a triple-mutated Fc variant in the best combination, and we elucidated the molecular interactions favoring Fc homodimer over heterodimer formation, which provided a structural basis for further optimization. The approach presented here demonstrates the feasibility of rational antibody modification for efficient and consistent production of monoclonal antibody mixtures in a single cell line and thus broadens our options for manufacturing more effective antibody-based therapeutic agents.

Solid ink-printed filter paper as a green adsorbent material for the solid-phase extraction of UV filters from water samples

ABSTRACTThis work describes the development of a new green solid-phase extraction approach, which is based on the use of low-cost extraction discs composed of plain filter papers that are covered with a synthetic wax-like coating. The filter papers are printed in a commercial solid ink printer, which dispenses a synthetic wax-like ink on the surface of the paper, to cover the hydrophilic cellulose fibre matrix with an interface of lipophilic domains where non-polar analytes can partition through hydrophobic interactions. The modified paper filters were used to extract hydrophobic organic compounds from water samples following the customary procedure of solid-phase extraction without sorbent preconditioning and needless of high-vacuum sources. As a proof-of-concept application, a series of non-polar organic UV filters were used as model analytes to optimise the extraction parameters and evaluate the performance of the method in spiked water samples. Based on this principle, a new sample preparation platform with low environmental footprint has been developed that enables extraction to be carried out using low-cost, environmental benign and non-toxic conventional materials. The advantages and disadvantages of the method, alongside with its future prospects towards the development of custom-made ‘printed extraction kits’, are envisioned and discussed.

Graphene-VP40 interactions and potential disruption of the Ebola virus matrix filaments

Ebola virus infections cause hemorrhagic fever that often results in very high fatality rates. In addition to exploring vaccines, development of drugs is also essential for treating the disease and preventing the spread of the infection. The Ebola virus matrix protein VP40 exists in various conformational and oligomeric forms and is a potential pharmacological target for disrupting the virus life-cycle. Here we explored graphene-VP40 interactions using molecular dynamics simulations and graphene pelleting assays. We found that graphene sheets associate strongly with VP40 at various interfaces. We also found that the graphene is able to disrupt the C-terminal domain (CTD-CTD) interface of VP40 hexamers. This VP40 hexamer-hexamer interface is crucial in forming the Ebola viral matrix and disruption of this interface may provide a method to use graphene or similar nanoparticle based solutions as a disinfectant that can significantly reduce the spread of the disease and prevent an Ebola epidemic.

Atomic‐Level Coupled Interfaces and Lattice Distortion on CuS/NiS2 Nanocrystals Boost Oxygen Catalysis for Flexible Zn‐Air Batteries

AbstractThe exploration of highly efficient nonprecious metal bifunctional electrocatalysts to boost oxygen evolution reaction and oxygen reduction reaction is critical for development of high energy density metal‐air batteries. Herein, a class of CuS/NiS2 interface nanocrystals (INs) catalysts with atomic‐level coupled nanointerface, subtle lattice distortion, and plentiful vacancy defects is reported. The results from temperature‐dependent in situ synchrotron‐based X‐ray absorption fine spectroscopy and electron spin resonance spectroscopy demonstrate that the lattice distortion of 14.7% in CuS/NiS2 caused by the strong Jahn–Teller effect of Cu, the strong atomic‐level coupled interface of CuS and NiS2 domains, and distinct vacancy defects can provide numerous effective active sites for their excellent bifunctional performance. A liquid Zn‐air battery with the CuS/NiS2 INs as air electrode displays a large peak power density (172.4 mW cm−2), a high specific capacity (775 mAh gZn−1), and long cycle life (up to 83 h), making the CuS/NiS2 INs among the best bifunctional catalysts for Zn‐air battery. More remarkably, the flexible CuS/NiS2 INs‐based solid‐state Zn‐air batteries can power the LED after twisting, making them be promising in portable and wearable electronic devices.

Conformationally constrained peptides target the allosteric kinase dimer interface and inhibit EGFR activation

Although EGFR is a highly sought-after drug target, inhibitor resistance remains a challenge. As an alternative strategy for kinase inhibition, we sought to explore whether allosteric activation mechanisms could effectively be disrupted. The kinase domain of EGFR forms an atypical asymmetric dimer via head-to-tail interactions and serves as a requisite for kinase activation. The kinase dimer interface is primarily formed by the H-helix derived from one kinase monomer and the small lobe of the second monomer. We hypothesized that a peptide designed to resemble the binding surface of the H-helix may serve as an effective disruptor of EGFR dimerization and activation. A library of constrained peptides was designed to mimic the H-helix of the kinase domain and interface side chains were optimized using molecular modeling. Peptides were constrained using peptide “stapling” to structurally reinforce an alpha-helical conformation. Peptide stapling was demonstrated to notably enhance cell permeation of an H-helix derived peptide termed EHBI2. Using cell-based assays, EHBI2 was further shown to significantly reduce EGFR activity as measured by EGFR phosphorylation and phosphorylation of the downstream signaling substrate Akt. To our knowledge, this is the first H-helix-based compound targeting the asymmetric interface of the kinase domain that can successfully inhibit EGFR activation and signaling. This study presents a novel, alternative targeting site for allosteric inhibition of EGFR.

Navigating the conformational landscape of G protein–coupled receptor kinases during allosteric activation

G protein-coupled receptors (GPCRs) are essential for transferring extracellular signals into carefully choreographed intracellular responses controlling diverse aspects of cell physiology. The duration of GPCR-mediated signaling is primarily regulated via GPCR kinase (GRK)-mediated phosphorylation of activated receptors. Although many GRK structures have been reported, the mechanisms underlying GRK activation are not well-understood, in part because it is unknown how these structures map to the conformational landscape available to this enzyme family. Unlike most other AGC kinases, GRKs rely on their interaction with GPCRs for activation and not phosphorylation. Here, we used principal component analysis of available GRK and protein kinase A crystal structures to identify their dominant domain motions and to provide a framework that helps evaluate how close each GRK structure is to being a catalytically competent state. Our results indicated that disruption of an interface formed between the large lobe of the kinase domain and the regulator of G protein signaling homology domain (RHD) is highly correlated with establishment of the active conformation. By introducing point mutations in the GRK5 RHD-kinase domain interface, we show with both in silico and in vitro experiments that perturbation of this interface leads to higher phosphorylation activity. Navigation of the conformational landscape defined by this bioinformatics-based study is likely common to all GPCR-activated GRKs.