Activated nanoscale actin-binding domain motion in the catenin-cadherin complex revealed by neutron spin echo spectroscopy.

As the core component of the adherens junction in cell-cell adhesion, the cadherin-catenin complex transduces mechanical tension between neighboring cells. Structural studies have shown that the cadherin-catenin complex exists as an ensemble of flexible conformations, with the actin-binding domain (ABD) of α-catenin adopting a variety of configurations. Here, we have determined the nanoscale protein domain dynamics of the cadherin-catenin complex using neutron spin echo spectroscopy (NSE), selective deuteration, and theoretical physics analyses. NSE reveals that, in the cadherin-catenin complex, the motion of the entire ABD becomes activated on nanosecond to submicrosecond timescales. By contrast, in the α-catenin homodimer, only the smaller disordered C-terminal tail of ABD is moving. Molecular dynamics (MD) simulations also show increased mobility of ABD in the cadherin-catenin complex, compared to the α-catenin homodimer. Biased MD simulations further reveal that the applied external forces promote the transition of ABD in the cadherin-catenin complex from an ensemble of diverse conformational states to specific states that resemble the actin-bound structure. The activated motion and an ensemble of flexible configurations of the mechanosensory ABD suggest the formation of an entropic trap in the cadherin-catenin complex, serving as negative allosteric regulation that impedes the complex from binding to actin under zero force. Mechanical tension facilitates the reduction in dynamics and narrows the conformational ensemble of ABD to specific configurations that are well suited to bind F-actin. Our results provide a protein dynamics and entropic explanation for the observed force-sensitive binding behavior of a mechanosensitive protein complex.

An ensemble of cadherin-catenin-vinculin complex employs vinculin as the major F-actin binding mode.

The cell-cell adhesion cadherin-catenin complexes recruit vinculin to the adherens junction (AJ) to modulate the mechanical couplings between neighboring cells. However, it is unclear how vinculin influences the AJ structure and function. Here, we identified two patches of salt bridges that lock vinculin in the head-tail autoinhibited conformation and reconstituted the full-length vinculin activation mimetics bound to the cadherin-catenin complex. The cadherin-catenin-vinculin complex contains multiple disordered linkers and is highly dynamic, which poses a challenge for structural studies. We determined the ensemble conformation of this complex using small-angle x-ray and selective deuteration/contrast variation small-angle neutron scattering. In the complex, both α-catenin and vinculin adopt an ensemble of flexible conformations, but vinculin has fully open conformations with the vinculin head and actin-binding tail domains well separated from each other. F-actin binding experiments show that the cadherin-catenin-vinculin complex binds and bundles F-actin. However, when the vinculin actin-binding domain is removed from the complex, only a minor fraction of the complex binds to F-actin. The results show that the dynamic cadherin-catenin-vinculin complex employs vinculin as the primary F-actin binding mode to strengthen AJ-cytoskeleton interactions.

An ensemble of flexible conformations underlies mechanotransduction by the cadherin-catenin adhesion complex.

The cadherin-catenin adhesion complex is the central component of the cell-cell adhesion adherens junctions that transmit mechanical stress from cell to cell. We have determined the nanoscale structure of the adherens junction complex formed by the α-catenin•ß-catenin•epithelial cadherin cytoplasmic domain (ABE) using negative stain electron microscopy, small-angle X-ray scattering, and selective deuteration/small-angle neutron scattering. The ABE complex is highly pliable and displays a wide spectrum of flexible structures that are facilitated by protein-domain motions in α- and ß-catenin. Moreover, the 107-residue intrinsically disordered N-terminal segment of ß-catenin forms a flexible "tongue" that is inserted into α-catenin and participates in the assembly of the ABE complex. The unanticipated ensemble of flexible conformations of the ABE complex suggests a dynamic mechanism for sensitivity and reversibility when transducing mechanical signals, in addition to the catch/slip bond behavior displayed by the ABE complex under mechanical tension. Our results provide mechanistic insight into the structural dynamics for the cadherin-catenin adhesion complex in mechanotransduction.

Dynamic structure of the full-length scaffolding protein NHERF1 influences signaling complex assembly.

The Na+/H+ exchange regulatory cofactor 1 (NHERF1) protein modulates the assembly and intracellular trafficking of several transmembrane G protein-coupled receptors (GPCRs) and ion transport proteins with the membrane-cytoskeleton adapter protein ezrin. Here, we applied solution NMR and small-angle neutron scattering (SANS) to structurally characterize full-length NHERF1 and disease-associated variants that are implicated in impaired phosphate homeostasis. Using NMR, we mapped the modular architecture of NHERF1, which is composed of two structurally-independent PDZ domains that are connected by a flexible, disordered linker. We observed that the ultra-long and disordered C-terminal tail of NHERF1 has a type 1 PDZ-binding motif that interacts weakly with the proximal, second PDZ domain to form a dynamically autoinhibited structure. Using ensemble-optimized analysis of SANS data, we extracted the molecular size distribution of structures from the extensive conformational space sampled by the flexible chain. Our results revealed that NHERF1 is a diffuse ensemble of variable PDZ domain configurations and a disordered C-terminal tail. The joint NMR/SANS data analyses of three disease variants (L110V, R153Q, and E225K) revealed significant differences in the local PDZ domain structures and in the global conformations compared with the WT protein. Furthermore, we show that the substitutions affect the affinity and kinetics of NHERF1 binding to ezrin and to a C-terminal peptide from G protein-coupled receptor kinase 6A (GRK6A). These findings provide important insight into the modulation of the intrinsic flexibility of NHERF1 by disease-associated point mutations that alter the dynamic assembly of signaling complexes.

α-Catenin Structure and Nanoscale Dynamics in Solution and in Complex with F-Actin.

As a core component of the adherens junction, α-catenin stabilizes the cadherin/catenin complexes to the actin cytoskeleton for the mechanical coupling of cell-cell adhesion. α-catenin also modulates actin dynamics, cell polarity, and cell-migration functions that are independent of the adherens junction. We have determined the solution structures of the α-catenin monomer and dimer using in-line size-exclusion chromatography small-angle X-ray scattering, as well as the structure of α-catenin dimer in complex to F-actin filament using selective deuteration and contrast-matching small angle neutron scattering. We further present the first observation, to our knowledge, of the nanoscale dynamics of α-catenin by neutron spin-echo spectroscopy, which explicitly reveals the mobile regions of α-catenin that are crucial for binding to F-actin. In solution, the α-catenin monomer is more expanded than either protomer shown in the crystal structure dimer, with the vinculin-binding M fragment and the actin-binding domain being able to adopt different configurations. The α-catenin dimer in solution is also significantly more expanded than the dimer crystal structure, with fewer interdomain and intersubunit contacts than the crystal structure. When in complex to F-actin, the α-catenin dimer has an even more open and extended conformation than in solution, with the actin-binding domain further separated from the main body of the dimer. The α-catenin-assembled F-actin bundle develops into an ordered filament packing arrangement at increasing α-catenin/F-actin molar ratios. Together, the structural and dynamic studies reveal that α-catenin possesses dynamic molecular conformations that prime this protein to function as a mechanosensor protein.

Origins of PDZ Binding Specificity. A Computational and Experimental Study Using NHERF1 and the Parathyroid Hormone Receptor.

Na+/H+ exchanger regulatory factor-1 (NHERF1) is a scaffolding protein containing two PSD95/discs large protein/ZO1 (PDZ) domains that modifies the signaling, trafficking, and function of the parathyroid hormone receptor (PTHR), a family B G-protein-coupled receptor. PTHR and NHERF1 bind through a PDZ-ligand-recognition mechanism. We show that PTH elicits phosphorylation of Thr591 in the canonical -ETVM binding motif of PTHR. Conservative substitution of Thr591 with Cys does not affect PTH(1-34)-induced cAMP production or binding of PTHR to NHERF1. The findings suggested the presence of additional sites upstream of the PDZ-ligand motif through which the two proteins interact. Structural determinants outside the canonical NHERF1 PDZ-PTHR interface that influence binding have not been characterized. We used molecular dynamics (MD) simulation to predict residues involved in these interactions. Simulation data demonstrate that the negatively charged Glu side chains at positions -3, -5, and -6 upstream of the PDZ binding motif are involved in PDZ-PTHR recognition. Engineered mutant peptides representing the PTHR C-terminal region were used to measure the binding affinity with NHERF1 PDZ domains. Comparable micromolar affinities for peptides of different length were confirmed by fluorescence polarization, isothermal titration calorimetry, and surface plasmon resonance. Binding affinities measured for Ala variants validate MD simulations. The linear relation between the change in enthalpy and entropy following Ala substitutions at upstream positions -3, -5, and -6 of the PTHR peptide provides a clear example of the thermodynamic compensation rule. Overall, our data highlight sequences in PTHR that contribute to NHERF1 interaction and can be altered to prevent phosphorylation-mediated inhibition.

Phosphatidylinositol 4,5-bisphosphate clusters the cell adhesion molecule CD44 and assembles a specific CD44-Ezrin heterocomplex, as revealed by small angle neutron scattering.

The cell adhesion molecule CD44 regulates diverse cellular functions, including cell-cell and cell-matrix interaction, cell motility, migration, differentiation, and growth. In cells, CD44 co-localizes with the membrane-cytoskeleton adapter protein Ezrin that links the CD44 assembled receptor signaling complexes to the cytoskeletal actin network, which organizes the spatial and temporal localization of signaling events. Here we report that the cytoplasmic tail of CD44 (CD44ct) is largely disordered. Upon binding to the signaling lipid phosphatidylinositol 4,5-bisphosphate (PIP2), CD44ct clusters into aggregates. Further, contrary to the generally accepted model, CD44ct does not bind directly to the FERM domain of Ezrin or to the full-length Ezrin but only forms a complex with FERM or with the full-length Ezrin in the presence of PIP2. Using contrast variation small angle neutron scattering, we show that PIP2 mediates the assembly of a specific heterotetramer complex of CD44ct with Ezrin. This study reveals the role of PIP2 in clustering CD44 and in assembling multimeric CD44-Ezrin complexes. We hypothesize that polyvalent electrostatic interactions are responsible for the assembly of CD44 clusters and the multimeric PIP2-CD44-Ezrin complexes.

Open conformation of ezrin bound to phosphatidylinositol 4,5-bisphosphate and to F-actin revealed by neutron scattering.

Ezrin is a member of the ezrin-radixin-moesin family (ERM) of adapter proteins that are localized at the interface between the cell membrane and the cortical actin cytoskeleton, and they regulate a variety of cellular functions. The structure representing a dormant and closed conformation of an ERM protein has previously been determined by x-ray crystallography. Here, using contrast variation small angle neutron scattering, we reveal the structural changes of the full-length ezrin upon binding to the signaling lipid phosphatidylinositol 4,5-bisphosphate (PIP(2)) and to F-actin. Ezrin binding to F-actin requires the simultaneous binding of ezrin to PIP(2). Once bound to F-actin, the opened ezrin forms more extensive contacts with F-actin than generally depicted, suggesting a possible role of ezrin in regulating the interfacial structure and dynamics between the cell membrane and the underlying actin cytoskeleton. In addition, using gel filtration, we find that the conformational opening of ezrin in response to PIP(2) binding is cooperative, but the cooperativity is disrupted by a phospho-mimic mutation S249D in the 4.1-ezrin/radixin/moesin (FERM) domain of ezrin. Using surface plasmon resonance, we show that the S249D mutation weakens the binding affinity and changes the kinetics of 4.1-ERM to PIP(2) binding. The study provides the first structural view of the activated ezrin bound to PIP(2) and to F-actin.

Nanoscale protein dynamics: a new frontier for neutron spin echo spectroscopy.

Recent studies show that neutron spin echo spectroscopy (NSE) can reveal long-range protein domain motions on nanometer lengthscales and on nanosecond to microsecond timescales. This unique capability of NSE provides new opportunities to understand protein dynamics and functions, such as how binding signals are propagated in a protein to distal sites. Here we review our applications of NSE to the study of nanoscale protein domain motions in a set of cell signaling proteins. We summarize the theoretical framework we have developed, which allows one to interpret the NSE data (Biophys. J. 99, 3473 (2010) and Proc. Natl. Acad. Sci. USA 102, 17646 (2005)). Our theoretical framework uses simple concepts from nonequilibrium statistical mechanics, and does not require elaborate molecular dynamics simulations, complex fits to rotational motion, or elastic network models. It is thus more robust than multiparameter techniques that require untestable assumptions. We also demonstrate our experimental scheme involving deuterium labeling of a protein domain or a subunit in a protein complex. We show that our selective deuteration scheme can highlight and resolve specific domain dynamics from the abundant global translational and rotational motions in a protein. Our approach thus clears significant hurdles to the application of NSE for the study of protein dynamics in solution.

A conformational switch in the scaffolding protein NHERF1 controls autoinhibition and complex formation.

The mammalian Na(+)/H(+) exchange regulatory factor 1 (NHERF1) is a multidomain scaffolding protein essential for regulating the intracellular trafficking and macromolecular assembly of transmembrane ion channels and receptors. NHERF1 consists of tandem PDZ-1, PDZ-2 domains that interact with the cytoplasmic domains of membrane proteins and a C-terminal (CT) domain that binds the membrane-cytoskeleton linker protein ezrin. NHERF1 is held in an autoinhibited state through intramolecular interactions between PDZ2 and the CT domain that also includes a C-terminal PDZ-binding motif (-SNL). We have determined the structures of the isolated and tandem PDZ2CT domains by high resolution NMR using small angle x-ray scattering as constraints. The PDZ2CT structure shows weak intramolecular interactions between the largely disordered CT domain and the PDZ ligand binding site. The structure reveals a novel helix-turn-helix subdomain that is allosterically coupled to the putative PDZ2 domain by a network of hydrophobic interactions. This helical subdomain increases both the stability and the binding affinity of the extended PDZ structure. Using NMR and small angle neutron scattering for joint structure refinement, we demonstrate the release of intramolecular domain-domain interactions in PDZ2CT upon binding to ezrin. Based on the structural information, we show that human disease-causing mutations in PDZ2, R153Q and E225K, have significantly reduced protein stability. Loss of NHERF1 expressed in cells could result in failure to assemble membrane complexes that are important for normal physiological functions.

Autoinhibitory interactions between the PDZ2 and C-terminal domains in the scaffolding protein NHERF1.

Na(+)/H(+) exchanger regulatory factor (NHERF1) is a signaling adaptor protein comprising two PDZ domains and a C-terminal ezrin-binding (EB) motif. To understand the role of intramolecular interactions in regulating its binding properties, we characterized the complex between the second PDZ domain PDZ2 and the C-terminal 242-358 fragment of NHERF1 using NMR and fluorescence methods. NMR chemical shift and relaxation data implicate 11 C-terminal residues in binding and, together with a thermodynamic analysis of mutant proteins, indicate that the EB region becomes helical when bound to PDZ2. Both specific contacts between PDZ2 and EB as well as nonspecific interactions involving a 100-residue flexible linker contribute to stabilizing two structurally distinct closed conformations of NHERF1. The affinity of mutant proteins for an extrinsic ligand is inversely related to the helix-forming propensity of the EB motif. The findings provide a structural framework for understanding how autoinhibitory interactions modulated the binding properties of NHERF1.

Activation of nanoscale allosteric protein domain motion revealed by neutron spin echo spectroscopy.

NHERF1 is a multidomain scaffolding protein that assembles signaling complexes, and regulates the cell surface expression and endocytic recycling of a variety of membrane proteins. The ability of the two PDZ domains in NHERF1 to assemble protein complexes is allosterically modulated by the membrane-cytoskeleton linker protein ezrin, whose binding site is located as far as 110 Ångstroms away from the PDZ domains. Here, using neutron spin echo (NSE) spectroscopy, selective deuterium labeling, and theoretical analyses, we reveal the activation of interdomain motion in NHERF1 on nanometer length-scales and on submicrosecond timescales upon forming a complex with ezrin. We show that a much-simplified coarse-grained model suffices to describe interdomain motion of a multidomain protein or protein complex. We expect that future NSE experiments will benefit by exploiting our approach of selective deuteration to resolve the specific domain motions of interest from a plethora of global translational and rotational motions. Our results demonstrate that the dynamic propagation of allosteric signals to distal sites involves changes in long-range coupled domain motions on submicrosecond timescales, and that these coupled motions can be distinguished and characterized by NSE.

Ligand-induced structural transitions in ErbB receptor extracellular domains.

Crystallographic studies showed that epidermal growth factor (EGF) receptor activation involves major domain rearrangements. Without bound ligand, the extracellular region of the receptor (sEGFR) adopts a "tethered" configuration with its dimerization site occluded by apparently autoinhibitory intramolecular interactions. Ligand binding causes the receptor to become "extended," breaking the tether and exposing the dimerization site. Using small-angle X-ray scattering (SAXS), we confirm that the tethered and extended conformations are also adopted in solution, and we describe low-resolution molecular envelopes for an intact sEGFR dimer. We also use SAXS to monitor directly the transition from a tethered to extended configuration in the monomeric extracellular regions of ErbB3 and a dimerization-defective EGFR mutant. Finally, we show that mutating every intramolecular tether interaction in sEGFR does not greatly alter its conformation. These findings explain why tether mutants fail to activate EGF receptor and provide new insight into regulation of ErbB receptor conformation.

Visualizing the nanoscale: protein internal dynamics and neutron spin echo spectroscopy.

The most complex molecular machines are proteins found within cells. Protein dynamics, in particular dynamics on nanoscales, presents us with a novel paradigm for cell signaling: the idea that proteins and protein complexes can communicate directly within themselves to effect long-range information transfer, via coupled domains and correlated residue clusters. This idea has been little explored, in large part because of a paucity of experimental techniques that can address the necessary questions. Here we review recent progress in developing a promising new approach, neutron spin echo spectroscopy.

Controllable Activation of Nanoscale Dynamics in a Disordered Protein Alters Binding Kinetics.

The phosphorylation of specific residues in a flexible disordered activation loop yields precise control of signal transduction. One paradigm is the phosphorylation of S339/S340 in the intrinsically disordered tail of the multi-domain scaffolding protein NHERF1, which affects the intracellular localization and trafficking of NHERF1 assembled signaling complexes. Using neutron spin echo spectroscopy (NSE), we show salt-concentration-dependent excitation of nanoscale motion at the tip of the C-terminal tail in the phosphomimic S339D/S340D mutant. The "tip of the whip" that is unleashed is near the S339/S340 phosphorylation site and flanks the hydrophobic Ezrin-binding motif. The kinetic association rate constant of the binding of the S339D/S340D mutant to the FERM domain of Ezrin is sensitive to buffer salt concentration, correlating with the excited nanoscale dynamics. The results suggest that electrostatics modulates the activation of nanoscale dynamics of an intrinsically disordered protein, controlling the binding kinetics of signaling partners. NSE can pinpoint the nanoscale dynamics changes in a highly specific manner.

Quantitative determination of the topological propensities of amyloidogenic peptides.

One of the interesting puzzles of amyloid beta-peptide of Alzheimer's disease (Abeta) is that it appears to polymerize into amyloid fibrils in a parallel beta sheet topology, while smaller subsets of the peptide produce anti-parallel beta sheets. In order to target potential weak points of amyloid fibrils in a rational drug design effort, it would be helpful to understand the forces that drive this change. We have designed two peptides CHQKLVFFAEDYNGKDEAFFVLKQHW and CHQKLVFFAEDYNGKHQKLVFFAEDW that join the significant amyloidogenic Abeta (14-23) sequence HQKLVFFAED in parallel and anti-parallel topologies, respectively. (Here, the word "parallel" refers only to residue sequence and not backbone topology). The N-termini of the hairpins were labeled with the fluorescent dye 5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid (IAEDANS), forming a fluorescence energy transfer donor-acceptor pair with the C-terminus tryptophan. Circular dichroism results show that the anti-parallel hairpin adopts a beta-sheet conformation, while the parallel hairpin is disordered. Fluorescent Resonance Energy Transfer (FRET) results show that the distance between the donor and the acceptor is significantly shorter in the anti-parallel topology than in the parallel topology. The fluorescence intensity of anti-parallel hairpin also displays a linear concentration dependence, indicating that the FRET observed in the anti-parallel hairpin is from intra-molecular interactions. The results thus provide a quantitative estimate of the relative topological propensities of amyloidogenic peptides. Our FRET and CD results show that beta sheets involving the essential Abeta (14-23) fragment, strongly prefer the anti-parallel topology. Moreover, we provide a quantitative estimate of the relative preference for these two topologies. Such analysis can be repeated for larger subsets of Abeta to determine quantitatively the relative degree of preference for parallel/anti-parallel topologies in given fragments of Abeta.

Essential Strategies for Revealing Nanoscale Protein Dynamics by Neutron Spin Echo Spectroscopy.

Determining the internal motions of a protein on nanosecond-to-microsecond timescales and on nanometer length scales is challenging by experimental biophysical techniques. Neutron spin echo spectroscopy (NSE) offers a unique opportunity to determine such nanoscale protein domain motions. However, the major hurdle in applying NSE to determine nanoscale protein motion is that the time and length scales of internal protein motions tend to be comparable to that of the global motions of a protein. The signals detected by NSE tend to be dominated by rigid-body translational and rotational diffusion. Using theoretical analyses, our laboratory showed that selective deuteration of a protein domain or a subunit can enhance the capability of NSE to reveal the internal motions in a protein complex. Here, we discuss the essential theoretical analysis and experimental methodology in detail. Protein nanomachines are far more complex than any molecular motors that have been artificially constructed, and their skillful utilization likely represents the future of medicine. With selective deuteration, NSE will allow us to see these nanomachines in motion.

Nucleotide binding induces changes in the oligomeric state and conformation of Sec A in a lipid environment: a small-angle neutron-scattering study.

In Escherichia coli, SecA is a large, multifunctional protein that is a vital component of the general protein secretion pathway. In its membrane-bound form it functions as the motor component of the protein translocase, perhaps through successive rounds of membrane insertion and ATP hydrolysis. To understand both the energy conversion process and translocase assembly, we have used contrast-matched, small-angle neutron-scattering (SANS) experiments to examine SecA in small unilamellar vesicles of E.coli phospholipids. In the absence of nucleotide, we observe a dimeric form of SecA with a radius of gyration comparable to that previously observed for SecA in solution. In contrast, the presence of either ADP or a non-hydrolyzable ATP analog induces conversion to a monomeric form. The larger radius of gyration for the ATP-bound relative to the ADP-bound form suggests the former has a more expanded global conformation. This is the first direct structural determination of SecA in a lipid bilayer. The SANS data indicate that nucleotide turnover can function as a switch of conformation of SecA in the membrane in a manner consistent with its proposed role in successive cycles of deep membrane penetration and release with concommitant preprotein insertion.

Nanoscale protein domain motion and long-range allostery in signaling proteins- a view from neutron spin echo sprectroscopy.

Many cellular proteins are multi-domain proteins. Coupled domain-domain interactions in these multidomain proteins are important for the allosteric relay of signals in the cellular signaling networks. We have initiated the application of neutron spin echo spectroscopy to the study of nanoscale protein domain motions on submicrosecond time scales and on nanometer length scale. Our NSE experiments reveal the activation of protein domain motions over a long distance of over more than 100 Å in a multidomain scaffolding protein NHERF1 upon binding to another protein Ezrin. Such activation of nanoscale protein domains motions is correlated with the allosteric assembly of multi-protein complexes by NHERF1 and Ezrin. Here, we summarize the theoretical framework that we have developed, which uses simple concepts from nonequilibrium statistical mechanics to interpret the NSE data, and employs a mobility tensor to describe nanoscale protein domain motion. Extracting nanoscale protein domain motion from the NSE does not require elaborate molecular dynamics simulations, or complex fits to rotational motion, or elastic network models. The approach is thus more robust than multiparameter techniques that require untestable assumptions. We also demonstrate that an experimental scheme of selective deuteration of a protein subunit in a complex can highlight and amplify specific domain dynamics from the abundant global translational and rotational motions in a protein. We expect NSE to provide a unique tool to determine nanoscale protein dynamics for the understanding of protein functions, such as how signals are propagated in a protein over a long distance to a distal domain.

Canonical and Noncanonical Sites Determine NPT2A Binding Selectivity to NHERF1 PDZ1.

Na+/H+ Exchanger Regulatory Factor-1 (NHERF1) is a scaffolding protein containing 2 PDZ domains that coordinates the assembly and trafficking of transmembrane receptors and ion channels. Most target proteins harboring a C-terminus recognition motif bind more-or-less equivalently to the either PDZ domain, which contain identical core-binding motifs. However some substrates such as the type II sodium-dependent phosphate co-transporter (NPT2A), uniquely bind only one PDZ domain. We sought to define the structural determinants responsible for the specificity of interaction between NHERF1 PDZ domains and NPT2A. By performing all-atom/explicit-solvent molecular dynamics (MD) simulations in combination with biological mutagenesis, fluorescent polarization (FP) binding assays, and isothermal titration calorimetry (ITC), we found that in addition to canonical interactions of residues at 0 and -2 positions, Arg at the -1 position of NPT2A plays a critical role in association with Glu43 and His27 of PDZ1 that are absent in PDZ2. Experimentally introduced mutation in PDZ1 (Glu43Asp and His27Asn) decreased binding to NPT2A. Conversely, introduction of Asp183Glu and Asn167His mutations in PDZ2 promoted the formation of favorable interactions yielding micromolar KDs. The results describe novel determinants within both the PDZ domain and outside the canonical PDZ-recognition motif that are responsible for discrimination of NPT2A between two PDZ domains. The results challenge general paradigms for PDZ recognition and suggest new targets for drug development.