Site-specific Spin Research Articles

Functional Conformational Changes in Lipoxygenase

Structures of catalytic domains of lipoxygenases, including representatives from mammals, corals, plants and pathogenic bacteria, are similar. All reveal a bent cavity passing close to the non-heme iron involved in catalysis. However, specificity for oxidation at one of several sites on the unsaturated fatty acid substrates lacks an overarching rationale. We have positioned spin labels at selected sites on helices-2, −9, −13, −15 and −22 in soybean lipoxygenase-1 and examined whether the substrate analog lysooleoyllecithin induces conformational changes at any of these sites. Site-specific spin labeling of introduced cysteines was achieved on a cysteine-free background in which each natural cysteine was substituted by serine. EPR spectra revealed conformational changes in helix-2 with lysolecithin binding and with pH variation, while the other sites did not. Site-directed spin labeling of helix-2 includes a spin-label scan of single sites for responses to lyso lecithin and also changes of charged residues in helix-2 to alanines to characterize the pH dependent changes. The spin label sites on the other helices are conformationally invariant and serve as the basis for ongoing of a lipoxygenase structure by paramagnetic distance geometry. All spin labeled lipoxygenase sites examined have enzymatic properties similar to native enzyme.

Rigid Core and Flexible Terminus: STRUCTURE OF SOLUBILIZED LIGHT-HARVESTING CHLOROPHYLL a/b COMPLEX (LHCII) MEASURED BY EPR

The structure of the major light-harvesting chlorophyll a/b complex (LHCII) was analyzed by pulsed EPR measurements and compared with the crystal structure. Site-specific spin labeling of the recombinant protein allowed the measurement of distance distributions over several intra- and intermolecular distances in monomeric and trimeric LHCII, yielding information on the protein structure and its local flexibility. A spin label rotamer library based on a molecular dynamics simulation was used to take the local mobility of spin labels into account. The core of LHCII in solution adopts a structure very similar or identical to the one seen in crystallized LHCII trimers with little motional freedom as indicated by narrow distance distributions along and between α helices. However, distances comprising the lumenal loop domain show broader distance distributions, indicating some mobility of this loop structure. Positions in the hydrophilic N-terminal domain, upstream of the first trans-membrane α helix, exhibit more and more mobility the closer they are to the N terminus. The nine amino acids at the very N terminus that have not been resolved in any of the crystal structure analyses give rise to very broad and possibly bimodal distance distributions, which may represent two families of preferred conformations.

The Subunit B-Dimer of the F1-ATPase Influences Conformational Transitions During Catalysis

The F1FO-ATP synthase, the enzyme responsible for most of ATP generated in biological systems, is a highly asymmetric enzyme complex. Part of the asymmetry stems from interactions of the nucleotide binding subunits α and β with the central rotor subunit gamma. Subunit gamma transfers the energy of the proton-driven rotation of the FO subunit c ring to subunit β, and induces conformational changes that drive ATP release during synthesis. We hypothesize that interactions with the external stalk subunit b-dimer will introduce additional asymmetry to the enzyme complex that may influence catalytic transitions. To investigate our hypothesis, we substituted two tyrosines in subunit β at amino acid positions 331 and 354 for cysteines in an otherwise cysteine-less enzyme. These tyrosines were shown previously to be part of the catalytic and non-catalytic sites, respectively. Using the cysteine-less enzyme as a control, our experiments showed that trapping efficiency of the cysteine-less protein as well as both mutants using AlFx- in what is considered to be a transition-state like conformation was influenced by the presence of soluble b2. Reactivation of trapped enzyme occurred in presence of excess of MgATP and was also influenced by the presence of soluble b2. In separate experiments using site-specific spin labeling and two different spin labels as reporter groups, we observed changes in the conformation of both the catalytic and non-catalytic sites during trapping. In each case, the presence of subunit b-dimer resulted in changes in the conformational transitions. Experiments are underway to localize the binding site of the subunit b-dimer in either the catalytic or the non-catalytic cleft of the ATPase.

Site-specific solvent exposure analysis of a membrane protein using unnatural amino acids and 19F nuclear magnetic resonance

Membrane proteins play an essential role in cellular metabolism, transportation and signal transduction across cell membranes. The scarcity of membrane protein structures has thus far prevented a full understanding of their molecular mechanisms. Preliminary topology studies and residue solvent exposure analysis have the potential to provide valuable information on membrane proteins of unknown structure. Here, a 19F-containing unnatural amino acid (trimethylfluoro-phenylalanine, tfmF) was applied to accomplish site-specific 19F spin incorporation at different sites in diacylglycerol kinase (DAGK, an Escherichia coli membrane protein) for site-specific solvent exposure analysis. Due to isotope effect on 19F spins, a standard curve for 19F-tfmF chemical shifts was drawn for varying solvent H2O/D2O ratios. Further site-specific 19F solvent isotope shift analysis was conducted for DAGK to distinguish residues in water-soluble loops, interfacial areas or hydrophobic membrane regions. This site-specific solvent exposure analysis method could be applied for further topological analysis of other membrane proteins.

Site-specific hydration dynamics in the nonpolar core of a molten globule by dynamic nuclear polarization of water.

Water-protein interactions play a direct role in protein folding. The chain collapse that accompanies protein folding involves extrusion of water from the nonpolar core. For many proteins, including apomyoglobin (apoMb), hydrophobic interactions drive an initial collapse to an intermediate state before folding to the final structure. However, the debate continues as to whether the core of the collapsed intermediate state is hydrated and, if so, what the dynamic nature of this water is. A key challenge is that protein hydration dynamics is significantly heterogeneous, yet suitable experimental techniques for measuring hydration dynamics with site-specificity are lacking. Here, we introduce Overhauser dynamic nuclear polarization at 0.35 T via site-specific nitroxide spin labels as a unique tool to probe internal and surface protein hydration dynamics with site-specific resolution in the molten globular, native, and unfolded protein states. The (1)H NMR signal enhancement of water carries information about the local dynamics of the solvent within ∼10 Å of a spin label. EPR is used synergistically to gain insights on local polarity and mobility of the spin-labeled protein. Several buried and solvent-exposed sites of apoMb are examined, each bearing a covalently bound nitroxide spin label. We find that the nonpoloar core of the apoMb molten globule is hydrated with water bearing significant translational dynamics, only 4-6-fold slower than that of bulk water. The hydration dynamics of the native state is heterogeneous, while the acid-unfolded state bears fast-diffusing hydration water. This study provides a high-resolution glimpse at the folding-dependent nature of protein hydration dynamics.

Measurement of Site-Specific 13C Spin−Lattice Relaxation in a Crystalline Protein

We demonstrate that it is possible to record site-specific spin-lattice relaxation rates for the majority of (13)C sites in uniformly (13)C and (15)N labeled solid proteins as a result of the slowing down of proton-driven spin diffusion at sample spinning frequencies > or = 60 kHz, thus providing a series of new experimental probes for characterizing molecular dynamics in solid proteins.

Site-specific electronic configurations of Fe 3d states by energy loss by channeled electrons

Site-specific configurations of Fe 3d electrons in a spinel ferrite were investigated by electron energy loss spectroscopy under electron channeling conditions. Site-specific spectra were extracted by applying a multivariate curve resolution (MCR) technique to the data set. An electronic difference in the Fe sites caused by ligand field splitting of trivalent Fe was probed. This demonstrated the promise of site-specific valence and spin state analysis in spintronics applications of spinel ferrites.

Structure of the Ternary Complex Formed by a Chemotaxis Receptor Signaling Domain, the CheA Histidine Kinase, and the Coupling Protein CheW As Determined by Pulsed Dipolar ESR Spectroscopy

The signaling apparatus that controls bacterial chemotaxis is composed of a core complex containing chemoreceptors, the histidine autokinase CheA, and the coupling protein CheW. Site-specific spin labeling and pulsed dipolar ESR spectroscopy (PDS) have been applied to investigate the structure of a soluble ternary complex formed by Thermotoga maritima CheA (TmCheA), CheW, and receptor signaling domains. Thirty-five symmetric spin-label sites (SLSs) were engineered into the five domains of the CheA dimer and CheW to provide distance restraints within the CheA:CheW complex in the absence and presence of a soluble receptor that inhibits kinase activity (Tm14). Additional PDS restraints among spin-labeled CheA, CheW, and an engineered single-chain receptor labeled at six different sites allow docking of the receptor structure relative to the CheA:CheW complex. Disulfide cross-linking between selectively incorporated Cys residues finds two pairs of positions that provide further constraints within the ternary complex: one involving Tm14 and CheW and another involving Tm14 and CheA. The derived structure of the ternary complex indicates a primary site of interaction between CheW and Tm14 that agrees well with previous biochemical and genetic data for transmembrane chemoreceptors. The PDS distance distributions are most consistent with only one CheW directly engaging one dimeric Tm14. The CheA dimerization domain (P3) aligns roughly antiparallel to the receptor-conserved signaling tip but does not interact strongly with it. The angle of the receptor axis with respect to P3 and the CheW-binding P5 domains is bound by two limits differing by approximately 20 degrees . In one limit, Tm14 aligns roughly along P3 and may interact to some extent with the hinge region near the P3 hairpin loop. In the other limit, Tm14 tilts to interact with the P5 domain of the opposite subunit in an interface that mimics that observed with the P5 homologue CheW. The time domain ESR data can be simulated from the model only if orientational variability is introduced for the P5 and, especially, P3 domains. The Tm14 tip also binds beside one of the CheA kinase domains (P4); however, in both bound and unbound states, P4 samples a broad range of distributions that are only minimally affected by Tm14 binding. The CheA P1 domains that contain the substrate histidine are also broadly distributed in space under all conditions. In the context of the hexagonal lattice formed by trimeric transmembrane chemoreceptors, the PDS structure is best accommodated with the P3 domain in the center of a honeycomb edge.

The Assembly of a Multisubunit Photosynthetic Membrane Protein Complex: A Site-Specific Spin Labeling EPR Spectroscopic Study of the PsaC Subunit in Photosystem I

The assembly of the PsaC subunit in the photosystem I (PS I) complex was studied using site-specific spin labeling electron paramagnetic resonance (EPR) spectroscopic techniques. The binding was monitored from the perspective of a reporter spin label attached to either the native C34(C) or the engineered C75(C) residue of wild-type PsaC (PsaC(WT)). Three distinct stages of PsaC assembly were analyzed: unbound PsaC, the P(700)-F(X)/PsaC complex, and the P(700)-F(X)/PsaC/PsaD complex. The changes in the EPR spectral line shape and the rotational correlation time of the spin label when PsaC(WT) binds to the PS I core are consistent with the conformational changes that are expected to occur during the assembly process. The addition of the PsaD subunit to the P(700)-F(X)/PsaC(WT-C34) complex induces further EPR spectral changes, which indicate that the presence of PsaD affects the orientation of the PsaC subunit on the PS I core. The binding of several PsaC variants, each lacking one or more key binding contacts with the PsaA/PsaB heterodimer, was monitored using a reporter spin label at C34(C). Our results indicate that the absence of the PsaC-PsaA/PsaB binding contacts causes PsaC to bind in an altered configuration on the PS I core. In particular, the removal of the entire C-terminus (PsaC(C-term)) causes PsaC to dock in a significantly different orientation when compared to the wild-type protein, as indicated by the EPR spectrum of the P(700)-F(X)/PsaC(C-term-C34) complex. Because the PsaC(C-term) variant retains only the symmetric network of PsaC-PsaA/PsaB ionic contacts, the altered EPR spectrum could, in principle, reflect a fraction of reaction centers that contain PsaC bound in the 180 degrees-rotated, C(2)-symmetry-related configuration. The results of this study are used to provide a comprehensive, stepwise mechanism for the binding of PsaC on the PS I core.

Probing the Structure of Membrane Proteins Using Pulsed EPR ESEEM Spectroscopy

Current methods used for determining the secondary structure of membrane proteins require large sample sizes and have long data acquisition times. Therefore using pulsed EPR spectroscopic technique of Electron Spin Echo Envelope Modulation (ESEEM) is advantageous due to the requirement of less sample, short data acquisition, and high sensitivity. Using this technique, we can determine short to medium range distances (up to 8 A) between a site-specific nitroxide spin label (MTSL) and a nearby NMR-active isotopic labeled residue for a variety of peptides and proteins which ultimately determine the difference between an α-helical and β-sheet secondary structure. The information can be obtained using a three-pulse ESEEM sequence which allows for the detection of the fundamental nuclear spin transitions. It is possible to calculate the radial distance between a paramagnetic electron and a weakly coupled nucleus because the modulation depth produced by a weakly dipolar-coupled nucleus is inversely proportional to the radial distance. This new method is applied to two different membrane peptides, M2δ AChR, and KIGAKI, which have α-helical and β-sheet structural components respectively. A nitroxide spin probe is attached to a specific Cys residue at a given position I, and a 2H isotopic labeled residue is attached to a nearby residue (i + 3). The corresponding data shows that an α-helical structure will yield a very large 2H peak, whereas a β-sheet will yield a much smaller 2H peak, thus a difference can be observed between the two secondary structures. The ESEEM technique requires less protein sample (about 75 μg) and data acquisition only takes about 1 hour which makes it a simple, powerful, and effective technique in the determination of the secondary structure of a membrane protein.

Investigating the Structural and Dynamic Properties of Membrane Proteins with Solid-State NMR, CW-EPR, ESEEM, and DEER Spectroscopic Techniques

Currently, we have very limited structural and dynamic information on membrane proteins and peptides. New spectroscopic methods are needed to probe these systems in a lipid bilayer. In order to address these issues, the Lorigan lab developing unique hybrid solid-state NMR and spin-label EPR spectroscopic techniques. Magnetic resonance spectroscopic data of 15N-, g2H-labeled and/or spin-labeled membrane proteins incorporated into vesicles and bicelles will be presented. State-of-the-art solid-state NMR and pulsed EPR techniques such as Magic Angle Spinning (MAS) NMR, Electron Spin Echo Envelope Modulation (ESEEM) spectroscopy, and Double Electron-Electron Resonance (DEER) spectroscopy will be used. The ESEEM technique can determine short to medium range distances (out to about 8 Å) between a site-specific nitroxide spin label (MTSL) and a nearby NMR-active isotopic labeled residue for a variety of different peptides and proteins which ultimately can be used to determine the difference between an α-helical and β-sheet secondary structure. DEER can be used to measure distances between 2 spin labels out to about 70 Å. The advantages and disadvantages of applying solid-state NMR and EPR spectroscopy to probe the structural and dynamic properties of membrane proteins will be discussed.

Self-Aggregation and Orientation of the Ion Channel-Forming Zervamicin IIA in the Membranes of ePC Vesicles Studied by cw EPR and ESEEM Spectroscopy

The ion channel-forming peptide antibiotic zervamicin A was studied in egg phosphocholin lipid membranes of large multilamellar vesicles (LMV) at 77 K. Continuous wave electron paramagnetic resonance (EPR) and electron spin echo envelope modulation (ESEEM) methods combined with site-specific electron spin labeling were used to study the aggregation and immersion depth of two analog molecules, i.e., each monolabeled either at the N- or C-terminal end of the helical molecule. Analysis of the shape of the EPR spectra indicates that zervamicin molecules form aggregates in which the dipolar interaction between the spin labels at the N-terminus is substantially larger than that between the labels at the C-terminus. The ESEEM method was used to study the interaction between the nitroxide radical spin labels of the zervamicin molecules and deuterium nuclei in LMV, which were prepared using a D2O buffer. It is established that the largest amplitude of deuterium modulation of the unpaired electron is observed for zervamicin molecules labeled at the N-terminus. Based on the analysis of the Fourier parameters of the deuterium modulated spectrum, a model of the immersion depth of the terminal ends of the zervamicin molecule in a lipid bilayer is formulated. All of the spin labels at the N-terminus are grouped at the lipid–water interface, whereas 60% of labels at the C-terminus are located at the lipid–water interface and 40% are more deeply inserted into the lipid bilayer.

High-field EPR

Among the numerous spectroscopic techniques utilized in photosynthesis research, high-field/high-frequency EPR and its pulse extensions ESE, ENDOR, ESEEM, and PELDOR play an important role in the endeavor to understand, on the basis of structure and dynamics data, dominant factors that control specificity and efficiency of light-induced electron- and proton-transfer processes in primary photosynthesis. Short-lived transient intermediates of the photocycle can be characterized by high-field EPR techniques, and detailed structural information can be obtained even from disordered sample preparations. The chapter describes how multifrequency high-field EPR methodology, in conjunction with mutation strategies for site-specific isotope or spin labeling and with the support of modern quantum-chemical computation methods for data interpretation, is capable of providing new insights into the photosynthetic transfer processes. The information obtained is complementary to that of protein crystallography, solid-state NMR and laser spectroscopy.

Dynamic nuclear polarization coupling factors calculated from molecular dynamics simulations of a nitroxide radical in water

The magnetic resonance signal obtained from nuclear spins is strongly affected by the presence of nearby electronic spins. This effect finds application in biomedical imaging and structural characterization of large biomolecules. In many of these applications nitroxide free radicals are widely used due to their non-toxicity and versatility as site-specific spin labels. We perform molecular dynamics simulations to study the electron-nucleus interaction of the nitroxide radical TEMPOL and water in atomistic detail. Correlation functions corresponding to the dipolar and scalar spin-spin couplings are computed from the simulations. The dynamic nuclear polarization coupling factors deduced from these correlation functions are in good agreement with experiment over a broad range of magnetic field strengths. The present approach can be applied to study solute-solvent interactions in general, and to characterize solvent dynamics on the surfaces of proteins or other spin-labeled biomolecules in particular.

Solution NMR Structure of Putidaredoxin–Cytochrome P450cam Complex via a Combined Residual Dipolar Coupling–Spin Labeling Approach Suggests a Role for Trp106 of Putidaredoxin in Complex Formation

The 58-kDa complex formed between the [2Fe–2S] ferredoxin, putidaredoxin (Pdx), and cytochrome P450cam (CYP101) from the bacterium Pseudomonas putida has been investigated by high-resolution solution NMR spectroscopy. Pdx serves as both the physiological reductant and effector for CYP101 in the enzymatic reaction involving conversion of substrate camphor to 5- exo-hydroxycamphor. In order to obtain an experimental structure for the oxidized Pdx–CYP101 complex, a combined approach using orientational data on the two proteins derived from residual dipolar couplings and distance restraints from site-specific spin labeling of Pdx has been applied. Spectral changes for residues in and near the paramagnetic metal cluster region of Pdx in complex with CYP101 have also been mapped for the first time using 15N and 13C NMR spectroscopy, leading to direct identification of the residues strongly affected by CYP101 binding. The new NMR structure of the Pdx–CYP101 complex agrees well with results from previous mutagenesis and biophysical studies involving residues at the binding interface such as formation of a salt bridge between Asp38 of Pdx and Arg112 of CYP101, while at the same time identifying key features different from those of earlier modeling studies. Analysis of the binding interface of the complex reveals that the side chain of Trp106, the C-terminal residue of Pdx and critical for binding to CYP101, is located across from the heme-binding loop of CYP101 and forms non-polar contacts with several residues in the vicinity of the heme group on CYP101, pointing to a potentially important role in complex formation.

Dynamic Nuclear Polarization Enhanced Nuclear Magnetic Resonance and Electron Spin Resonance Studies of Hydration and Local Water Dynamics in Micelle and Vesicle Assemblies

We present a unique analysis tool for the selective detection of local water inside soft molecular assemblies (hydrophobic cores, vesicular bilayers, and micellar structures) suspended in bulk water. Through the use of dynamic nuclear polarization (DNP), the (1)H NMR signal of water is amplified, as it interacts with stable radicals that possess approximately 658 times higher spin polarization. We utilized stable nitroxide radicals covalently attached along the hydrophobic tail of stearic acid molecules that incorporate themselves into surfactant-based micelle or vesicle structures. Here, we present a study of local water content and fluid viscosity inside oleate micelles and vesicles and Triton X-100 micelles to serve as model systems for soft molecular assemblies. This approach is unique because the amplification of the NMR signal is performed in bulk solution and under ambient conditions with site-specific spin labels that only detect the water that is directly interacting with the localized spin labels. Continuous wave (cw) electron spin resonance (ESR) analysis provides rotational dynamics of the spin-labeled molecular chain segments and local polarity parameters that can be related to hydration properties, whereas we show that DNP-enhanced (1)H NMR analysis of fluid samples directly provides translational water dynamics and permeability of the local environment probed by the spin label. Our technique therefore has the potential to become a powerful analysis tool, complementary to cw ESR, to study hydration characteristics of surfactant assemblies, lipid bilayers, or protein aggregates, where water dynamics is a key parameter of their structure and function. In this study, we find that there is significant penetration of water inside the oleate micelles with a higher average local water viscosity (approximately 1.8 cP) than in bulk water, and Triton X-100 micelles and oleate vesicle bilayers mostly exclude water while allowing for considerable surfactant chain motion and measurable water permeation through the soft structure.

Role of Sequence Bias in the Topology of the Multidrug Transporter EmrE

EmrE is the prototype of small multidrug resistance transporters and has emerged as a model of membrane protein evolution. Analysis of the distances separating symmetry-related site-specific spin labels, correlation of topological sequence bias to C-terminal orientation, to membrane insertion efficiency, and to resistance to ethidium bromide collectively demonstrate that EmrE monomers adopt a parallel topology in the functional dimer. We propose a coupled insertion and assembly model for EmrE in which the favorable energetics of the parallel dimer interface override topological constraints arising from weak asymmetry in positive charge distribution.

S1.13 The external stalk of the FOF1F0F1-ATPase: Three-dimensional structure of the subunit b-dimer as determined by site-specific spin labeling, ESR and molecular modeling

S1.13 The external stalk of the FOF1F0F1-ATPase: Three-dimensional structure of the subunit b-dimer as determined by site-specific spin labeling, ESR and molecular modeling

Determination of the Oligomeric States of Human and Rat Monoamine Oxidases in the Outer Mitochondrial Membrane and Octyl β-d-Glucopyranoside Micelles Using Pulsed Dipolar Electron Spin Resonance Spectroscopy

Human monoamine oxidase A (hMAOA) is considered to be unique among mammalian MAOs in having a non-conservative Glu-X-Lys mutation (X being 151 in MAOAs and 142 in MAOB's), which is suggested to be the reason for its monomeric structure. This hypothesis has been tested in this work. A pargyline based nitroxide spin labeled irreversible inhibitor (ParSL) was used as a MAO active site specific spin probe to measure intersubunit distances in detergent (octyl beta-d-glucopyranoside, OGP) purified and OMM bound forms by a pulsed dipolar ESR spectroscopic (PDS) technique. In a parallel approach, the covalent flavin cofactor present in the MAO active sites was reduced to its respective anionic flavin semiquinone and used for measuring inter-flavin distances in detergent purified samples. The measured interspin distances are within 0.1-0.3 nm of those estimated from the available dimeric crystal structures of human MAOB and rat MAOA and show that all human and rat MAOs exist as dimers in the OMM. In the OGP micelle, however, human and rat MAOAs exist only partially (<or=50%) as dimers, whereas human and rat MAOBs exist entirely as dimers. The Lys-151-Glu mutant of human MAOA and the Glu-142-Lys mutant of human MAOB exhibit similar spectral properties as the corresponding wild-type enzymes. Therefore, no role of the Glu-X residue in stabilizing dimeric structures of MAOs was found. The monomeric crystal structure reported for human MAOA is thus a result of its instability in the OGP micelles. The general applicability of the PDS technique to structural studies of membrane proteins in their native membrane environments and detergent purified forms is discussed.

Mechanism of Constitutive Phosphoinositide 3-Kinase Activation by Oncogenic Mutants of the p85 Regulatory Subunit

p85/p110 phosphoinositide 3-kinases regulate multiple cell functions and are frequently mutated in human cancer. The p85 regulatory subunit stabilizes and inhibits the p110 catalytic subunit. The minimal fragment of p85 capable of regulating p110 is the N-terminal SH2 domain linked to the coiled-coil iSH2 domain (referred to as p85ni). We have previously proposed that the conformationally rigid iSH2 domain tethers p110 to p85, facilitating regulatory interactions between p110 and the p85 nSH2 domain. In an oncogenic mutant of murine p85, truncation at residue 571 leads to constitutively increased phosphoinositide 3-kinase activity, which has been proposed to result from either loss of an inhibitory Ser-608 autophosphorylation site or altered interactions with cellular regulatory factors. We have examined this mutant (referred to as p65) in vitro and find that p65 binds but does not inhibit p110, leading to constitutive p110 activity. This activated phenotype is observed with recombinant proteins in the absence of cellular factors. Importantly, this effect is also produced by truncating p85ni at residue 571. Thus, the phenotype is not because of loss of the Ser-608 inhibitory autophosphorylation site, which is not present in p85ni. To determine the structural basis for the phenotype of p65, we used a broadly applicable spin label/NMR approach to define the positioning of the nSH2 domain relative to the iSH2 domain. We found that one face of the nSH2 domain packs against the 581-593 region of the iSH2 domain. The loss of this interaction in the truncated p65 would remove the orienting constraints on the nSH2 domain, leading to a loss of p110 regulation by the nSH2. Based on these findings, we propose a general model for oncogenic mutants of p85 and p110 in which disruption of nSH2-p110 regulatory contacts leads to constitutive p110 activity.