Structure of a 28.5 kDa duplex-embedded G-quadruplex system resolved to 7.4 Å resolution with cryo-EM.

Genomic regions with high guanine content can fold into non-B form DNA four-stranded structures known as G-quadruplexes (G4s). Extensive in vivo investigations have revealed that promoter G4s are transcriptional regulators. Little structural information exists for these G4s embedded within duplexes, their presumed genomic environment. Here, we report the 7.4 Å resolution structure and dynamics of a 28.5 kDa duplex-G4-duplex (DGD) model system using cryo-EM, molecular dynamics, and small-angle X-ray scattering (SAXS) studies. The DGD cryo-EM refined model features a 53° bend induced by a stacked duplex-G4 interaction at the 5' G-tetrad interface with a persistently unstacked 3' duplex. The surrogate complement poly dT loop preferably stacks onto the 3' G-tetrad interface resulting in occlusion of both 5' and 3' tetrad interfaces. Structural analysis shows that the DGD model is quantifiably more druggable than the monomeric G4 structure alone and represents a new structural drug target. Our results illustrate how the integration of cryo-EM, MD, and SAXS can reveal complementary detailed static and dynamic structural information on DNA G4 systems.

Structural and functional studies reveal the molecular basis of substrate promiscuity of a glycosyltransferase originating from a major agricultural pest.

The two-spotted spider mite, Tetranychus urticae, is a major cosmopolitan pest that feeds on more than 1100 plant species. Its genome contains an unprecedentedly large number of genes involved in detoxifying and transporting xenobiotics, including 80 genes that code for UDP glycosyltransferases (UGTs). These enzymes were acquired via horizontal gene transfer from bacteria after loss in the Chelicerata lineage. UGTs are well-known for their role in phase II metabolism; however, their contribution to host adaptation and acaricide resistance in arthropods, such as T. urticae, is not yet resolved. TuUGT202A2 (Tetur22g00270) has been linked to the ability of this pest to adapt to tomato plants. Moreover, it was shown that this enzyme can glycosylate a wide range of flavonoids. To understand this relationship at the molecular level, structural, functional, and computational studies were performed. Structural studies provided specific snapshots of the enzyme in different catalytically relevant stages. The crystal structure of TuUGT202A2 in complex with UDP-glucose was obtained and site-directed mutagenesis paired with molecular dynamic simulations revealed a novel lid-like mechanism involved in the binding of the activated sugar donor. Two additional TuUGT202A2 crystal complexes, UDP-(S)-naringenin and UDP-naringin, demonstrated that this enzyme has a highly plastic and open-ended acceptor-binding site. Overall, this work reveals the molecular basis of substrate promiscuity of TuUGT202A2 and provides novel insights into the structural mechanism of UGTs catalysis.

Mechanistic insights into enhancement or inhibition of phase separation by different polyubiquitin chains.

Ubiquitin-binding shuttle UBQLN2 mediates crosstalk between proteasomal degradation and autophagy, likely via interactions with K48- and K63-linked polyubiquitin chains, respectively. UBQLN2 comprises self-associating regions that drive its homotypic liquid-liquid phase separation (LLPS). Specific interactions between one of these regions and ubiquitin inhibit UBQLN2 LLPS. Here, we show that, unlike ubiquitin, the effects of multivalent polyubiquitin chains on UBQLN2 LLPS are highly dependent on chain types. Specifically, K11-Ub4 and K48-Ub4 chains generally inhibit UBQLN2 LLPS, whereas K63-Ub4, M1-Ub4 chains, and a designed tetrameric ubiquitin construct significantly enhance LLPS. We demonstrate that these opposing effects stem from differences in chain conformations but not in affinities between chains and UBQLN2. Chains with extended conformations and increased accessibility to the ubiquitin-binding surface promote UBQLN2 LLPS by enabling a switch between homotypic to partially heterotypic LLPS that is driven by both UBQLN2 self-interactions and interactions between multiple UBQLN2 units with each polyubiquitin chain. Our study provides mechanistic insights into how the structural and conformational properties of polyubiquitin chains contribute to heterotypic LLPS with ubiquitin-binding shuttles and adaptors.

Homomeric interactions of the MPZ Ig domain and their relation to Charcot-Marie-Tooth disease.

Mutations in MPZ (myelin protein zero) can cause demyelinating early-onset Charcot-Marie-Tooth type 1B disease or later onset type 2I/J disease characterized by axonal degeneration, reflecting the diverse roles of MPZ in Schwann cells. MPZ holds apposing membranes of the myelin sheath together, with the adhesion role fulfilled by its extracellular immunoglobulin-like domain (IgMPZ), which oligomerizes. Models for how the IgMPZ might form oligomeric assemblies has been extrapolated from a protein crystal structure in which individual rat IgMPZ subunits are packed together under artificial conditions, forming three weak interfaces. One interface organizes the IgMPZ into tetramers, a second 'dimer' interface links tetramers together across the intraperiod line, and a third hydrophobic interface that mediates binding to lipid bilayers or the same hydrophobic surface on another IgMPZ domain. Presently, there are no data confirming whether the proposed IgMPZ interfaces actually mediate oligomerization in solution, whether they are required for the adhesion activity of MPZ, whether they are important for myelination, or whether their loss results in disease. We performed nuclear magnetic resonance spectroscopy and small angle X-ray scattering analysis of wild-type IgMPZ as well as mutant forms with amino acid substitutions designed to interrupt its presumptive oligomerization interfaces. Here, we confirm the interface that mediates IgMPZ tetramerization, but find that dimerization is mediated by a distinct interface that has yet to be identified. We next correlated different types of Charcot-Marie-Tooth disease symptoms to subregions within IgMPZ tetramers. Variants causing axonal late-onset disease (CMT2I/J) map to surface residues of IgMPZ proximal to the transmembrane domain. Variants causing early-onset demyelinating disease (CMT1B) segregate into two groups: one is described by variants that disrupt the stability of the Ig-fold itself and are largely located within the core of the IgMPZ domain; whereas another describes a region on the surface of IgMPZ tetramers, accessible to protein interactions. Computational docking studies predict that this latter disease-relevant subregion may potentially mediate dimerization of IgMPZ tetramers.

Long promoter sequences form higher-order G-quadruplexes: an integrative structural biology study of c-Myc, k-Ras and c-Kit promoter sequences.

We report on higher-order G-quadruplex structures adopted by long promoter sequences obtained by an iterative integrated structural biology approach. Our approach uses quantitative biophysical tools (analytical ultracentrifugation, small-angle X-ray scattering, and circular dichroism spectroscopy) combined with modeling and molecular dynamics simulations, to derive self-consistent structural models. The formal resolution of our approach is 18 angstroms, but in some cases structural features of only a few nucleotides can be discerned. We report here five structures of long (34-70 nt) wild-type sequences selected from three cancer-related promoters: c-Myc, c-Kit and k-Ras. Each sequence studied has a unique structure. Three sequences form structures with two contiguous, stacked, G-quadruplex units. One longer sequence from c-Myc forms a structure with three contiguous stacked quadruplexes. A longer c-Kit sequence forms a quadruplex-hairpin structure. Each structure exhibits interfacial regions between stacked quadruplexes or novel loop geometries that are possible druggable targets. We also report methodological advances in our integrated structural biology approach, which now includes quantitative CD for counting stacked G-tetrads, DNaseI cleavage for hairpin detection and SAXS model refinement. Our results suggest that higher-order quadruplex assemblies may be a common feature within the genome, rather than simple single quadruplex structures.

Sas20 is a highly flexible starch-binding protein in the Ruminococcus bromii cell-surface amylosome.

Ruminococcus bromii is a keystone species in the human gut that has the rare ability to degrade dietary resistant starch (RS). This bacterium secretes a suite of starch-active proteins that work together within larger complexes called amylosomes that allow R. bromii to bind and degrade RS. Starch adherence system protein 20 (Sas20) is one of the more abundant proteins assembled within amylosomes, but little could be predicted about its molecular features based on amino acid sequence. Here, we performed a structure-function analysis of Sas20 and determined that it features two discrete starch-binding domains separated by a flexible linker. We show that Sas20 domain 1 contains an N-terminal ß-sandwich followed by a cluster of α-helices, and the nonreducing end of maltooligosaccharides can be captured between these structural features. Furthermore, the crystal structure of a close homolog of Sas20 domain 2 revealed a unique bilobed starch-binding groove that targets the helical α1,4-linked glycan chains found in amorphous regions of amylopectin and crystalline regions of amylose. Affinity PAGE and isothermal titration calorimetry demonstrated that both domains bind maltoheptaose and soluble starch with relatively high affinity (Kd ≤ 20 µM) but exhibit limited or no binding to cyclodextrins. Finally, small-angle X-ray scattering analysis of the individual and combined domains support that these structures are highly flexible, which may allow the protein to adopt conformations that enhance its starch-targeting efficiency. Taken together, we conclude that Sas20 binds distinct features within the starch granule, facilitating the ability of R. bromii to hydrolyze dietary RS.

The Disordered Amino Terminus of the Circadian Enzyme Nocturnin Modulates Its NADP(H) Phosphatase Activity by Changing Protein Dynamics.

Endogenous circadian clocks control the rhythmicity of a broad range of behavioral and physiological processes, and this is entrained by the daily fluctuations in light and dark. Nocturnin (Noct) is a rhythmically expressed gene regulated by the circadian clock that belongs to the CCR4 family of endonuclease-exonuclease-phosphatase (EEP) enzymes, and the NOCT protein exhibits phosphatase activity, catalyzing the removal of the 2'-phosphate from NADP(H). In addition to its daily nighttime peak of expression, it is also induced by acute stimuli. Loss of Nocturnin (Noct-/-) in mice results in resistance to high-fat diet-induced obesity, and loss of Noct in HEK293T cells confers a protective effect to oxidative stress. Modeling of the full-length Nocturnin protein reveals a partially structured amino terminus that is disparate from its CCR4 family members. The high sequence conservation of a leucine zipper-like (LZ-like) motif, the only structural element in the amino terminus, highlights the potential importance of this domain in modulating phosphatase activity. In vitro biochemical and biophysical techniques demonstrate that the LZ-like domain within the flexible N-terminus is necessary for preserving the active site cleft in an optimal conformation to promote the efficient turnover of the substrate. This modulation occurs in cis and is pivotal in maintaining the stability and conformational integrity of the enzyme. These new findings suggest an additional layer of modulating the activity of Nocturnin in addition to its rhythmicity to provide fine-tuned control over cellular levels of NADPH.

Functional and structural characterization of allosteric activation of phospholipase Cε by Rap1A.

Phospholipase Cε (PLCε) is activated downstream of G protein-coupled receptors and receptor tyrosine kinases through direct interactions with small GTPases, including Rap1A and Ras. Although Ras has been reported to allosterically activate the lipase, it is not known whether Rap1A has the same ability or what its molecular mechanism might be. Rap1A activates PLCε in response to the stimulation of ß-adrenergic receptors, translocating the complex to the perinuclear membrane. Because the C-terminal Ras association (RA2) domain of PLCε was proposed to the primary binding site for Rap1A, we first confirmed using purified proteins that the RA2 domain is indeed essential for activation by Rap1A. However, we also showed that the PLCε pleckstrin homology (PH) domain and first two EF hands (EF1/2) are required for Rap1A activation and identified hydrophobic residues on the surface of the RA2 domain that are also necessary. Small-angle X-ray scattering showed that Rap1A binding induces and stabilizes discrete conformational states in PLCε variants that can be activated by the GTPase. These data, together with the recent structure of a catalytically active fragment of PLCε, provide the first evidence that Rap1A, and by extension Ras, allosterically activate the lipase by promoting and stabilizing interactions between the RA2 domain and the PLCε core.

The first DEP domain of the RhoGEF P-Rex1 autoinhibits activity and contributes to membrane binding.

Phosphatidylinositol (3,4,5)-trisphosphate (PIP3)-dependent Rac exchanger 1 (P-Rex1) catalyzes the exchange of GDP for GTP on Rac GTPases, thereby triggering changes in the actin cytoskeleton and in transcription. Its overexpression is highly correlated with the metastasis of certain cancers. P-Rex1 recruitment to the plasma membrane and its activity are regulated via interactions with heterotrimeric Gßγ subunits, PIP3, and protein kinase A (PKA). Deletion analysis has further shown that domains C-terminal to its catalytic Dbl homology (DH) domain confer autoinhibition. Among these, the first dishevelled, Egl-10, and pleckstrin domain (DEP1) remains to be structurally characterized. DEP1 also harbors the primary PKA phosphorylation site, suggesting that an improved understanding of this region could substantially increase our knowledge of P-Rex1 signaling and open the door to new selective chemotherapeutics. Here we show that the DEP1 domain alone can autoinhibit activity in context of the DH/PH-DEP1 fragment of P-Rex1 and interacts with the DH/PH domains in solution. The 3.1 Å crystal structure of DEP1 features a domain swap, similar to that observed previously in the Dvl2 DEP domain, involving an exposed basic loop that contains the PKA site. Using purified proteins, we show that although DEP1 phosphorylation has no effect on the activity or solution conformation of the DH/PH-DEP1 fragment, it inhibits binding of the DEP1 domain to liposomes containing phosphatidic acid. Thus, we propose that PKA phosphorylation of the DEP1 domain hampers P-Rex1 binding to negatively charged membranes in cells, freeing the DEP1 domain to associate with and inhibit the DH/PH module.

Biophysical Characterization of Cancer-Related Carbonic Anhydrase IX.

Upregulation of carbonic anhydrase IX (CA IX) is associated with several aggressive forms of cancer and promotes metastasis. CA IX is normally constitutively expressed at low levels in selective tissues associated with the gastrointestinal tract, but is significantly upregulated upon hypoxia in cancer. CA IX is a multi-domain protein, consisting of a cytoplasmic region, a single-spanning transmembrane helix, an extracellular CA catalytic domain, and a proteoglycan-like (PG) domain. Considering the important role of CA IX in cancer progression and the presence of the unique PG domain, little information about the PG domain is known. Here, we report biophysical characterization studies to further our knowledge of CA IX. We report the 1.5 Å resolution crystal structure of the wild-type catalytic domain of CA IX as well as small angle X-ray scattering and mass spectrometry of the entire extracellular region. We used matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry to characterize the spontaneous degradation of the CA IX PG domain and confirm that it is only the CA IX catalytic domain that forms crystals. Small angle X-ray scattering analysis of the intact protein indicates that the PG domain is not randomly distributed and adopts a compact distribution of shapes in solution. The observed dynamics of the extracellular domain of CA IX could have physiological relevance, including observed cleavage and shedding of the PG domain.

Temperature-dependent radiation sensitivity and order of 70S ribosome crystals.

All evidence to date indicates that at T = 100 K all protein crystals exhibit comparable sensitivity to X-ray damage when quantified using global metrics such as change in scaling B factor or integrated intensity versus dose. This is consistent with observations in cryo-electron microscopy, and results because nearly all diffusive motions of protein and solvent, including motions induced by radiation damage, are frozen out. But how do the sensitivities of different proteins compare at room temperature, where radiation-induced radicals are free to diffuse and protein and lattice structures are free to relax in response to local damage? It might be expected that a large complex with extensive conformational degrees of freedom would be more radiation sensitive than a small, compact globular protein. As a test case, the radiation sensitivity of 70S ribosome crystals has been examined. At T = 100 and 300 K, the half doses are 64 MGy (at 3 Šresolution) and 150 kGy (at 5 Šresolution), respectively. The maximum tolerable dose in a crystallography experiment depends upon the initial or desired resolution. When differences in initial data-set resolution are accounted for, the former half dose is roughly consistent with that for model proteins, and the 100/300 K half-dose ratio is roughly a factor of ten larger. 70S ribosome crystals exhibit substantially increased resolution at 100 K relative to 300 K owing to cooling-induced ordering and not to reduced radiation sensitivity and slower radiation damage.

BioXTAS RAW 2: new developments for a free open-source program for small-angle scattering data reduction and analysis.

BioXTAS RAW is a free open-source program for reduction, analysis and modelling of biological small-angle scattering data. Here, the new developments in RAW version 2 are described. These include improved data reduction using pyFAI; updated automated Guinier fitting and D max finding algorithms; automated series (e.g. size-exclusion chromatography coupled small-angle X-ray scattering or SEC-SAXS) buffer- and sample-region finding algorithms; linear and integral baseline correction for series; deconvolution of series data using regularized alternating least squares (REGALS); creation of electron-density reconstructions using electron density via solution scattering (DENSS); a comparison window showing residuals, ratios and statistical comparisons between profiles; and generation of PDF reports with summary plots and tables for all analysis. Furthermore, there is now a RAW API, which can be used without the graphical user interface (GUI), providing full access to all of the functionality found in the GUI. In addition to these new capabilities, RAW has undergone significant technical updates, such as adding Python 3 compatibility, and has entirely new documentation available both online and in the program.

Invariant BECN1 CXXC motifs bind Zn2+ and regulate structure and function of the BECN1 intrinsically disordered region.

ABBREVIATIONS: AFM: aromatic finger mutant; BH3D: BCL2 homology 3 domain; CCD: coiled-coil domain; CD: circular dichroism spectroscopy; [CysDM1]: C18S and C21S double mutant; [CysDM2]: C137S, and C140S double mutant; [CysTM], C18S, C21S, C137S, and C140S tetrad mutant; Dmax: maximum particle diameter; dRI, differential refractive index; EFA: evolving factor analysis; FHD: flexible helical domain; FL: full length; GFP: green fluorescent protein; HDX-MS: hydrogen/deuterium exchange mass spectrometry; ICP-MS: inductively coupled plasma mass spectrometry; IDR: intrinsically disordered region; ITC, isothermal titration calorimetry; MALS, multi angle light scattering; MBP: maltose-binding protein; MoRFs: molecular recognition features; P(r): pairwise-distance distribution; PtdIns3K: class III phosphatidylinositol 3-kinase; Rg: radius of gyration; SASBDB: small angle scattering biological data bank; SEC: size-exclusion chromatography; SEC-SAXS: size-exclusion chromatography in tandem with small angle X-ray scattering; TEV: tobacco-etch virus; TFE: 2,2,2-trifluoroethanol; TPEN: N,N,N,N-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine; Vc: volume of correlation; WT: wild-type.

Structural insights into the formation of repulsive netrin guidance complexes.

Netrins dictate attractive and repulsive responses during axon growth and cell migration, where the presence of the receptor Uncoordinated-5 (UNC-5) on target cells results in repulsion. Here, we showed that UNC-5 is a heparin-binding protein, determined its structure bound to a heparin fragment, and could modulate UNC-5-heparin affinity using a directed evolution platform or structure-based rational design. We demonstrated that UNC-5 and UNC-6/netrin form a large, stable, and rigid complex in the presence of heparin, and heparin and UNC-5 exclude the attractive UNC-40/DCC receptor from binding to UNC-6/netrin to a large extent. Caenorhabditis elegans with a heparin-binding-deficient UNC-5 fail to establish proper gonad morphology due to abrogated cell migration, which relies on repulsive UNC-5 signaling in response to UNC-6. Combining UNC-5 mutations targeting heparin and UNC-6/netrin contacts results in complete cell migration and axon guidance defects. Our findings establish repulsive netrin responses to be mediated through a glycosaminoglycan-regulated macromolecular complex.

A General Mechanism for the General Stress Response in Bacteria.

The General Stress Response promotes survival of bacteria in adverse conditions, but how sensor proteins transduce species-specific signals to initiate the response is not known. The serine/threonine phosphatase RsbU initiates the General Stress Response in B. subtilis upon binding a partner protein (RsbT) that is released from sequestration by environmental stresses. We report that RsbT activates RsbU by inducing otherwise flexible linkers of RsbU to form a short coiled-coil that dimerizes and activates the phosphatase domains. Importantly, we present evidence that related coiled-coil linkers and phosphatase dimers transduce signals from diverse sensor domains to control the General Stress Response and other signaling across bacterial phyla. These results additionally resolve the mystery of how shared sensory domains control serine/threonine phosphatases, diguanylate cyclases and histidine kinases, revealing a common coiled-coil linker transduction mechanism. We propose that this provides bacteria with a modularly exchangeable toolkit for the evolution of diverse signaling pathways.

The Ruminococcus bromii amylosome protein Sas6 binds single and double helical α-glucan structures in starch.

Resistant starch is a prebiotic accessed by gut bacteria with specialized amylases and starch-binding proteins. The human gut symbiont Ruminococcus bromii expresses Sas6 (Starch Adherence System member 6), which consists of two starch-specific carbohydrate-binding modules from family 26 (RbCBM26) and family 74 (RbCBM74). Here, we present the crystal structures of Sas6 and of RbCBM74 bound with a double helical dimer of maltodecaose. The RbCBM74 starch-binding groove complements the double helical α-glucan geometry of amylopectin, suggesting that this module selects this feature in starch granules. Isothermal titration calorimetry and native mass spectrometry demonstrate that RbCBM74 recognizes longer single and double helical α-glucans, while RbCBM26 binds short maltooligosaccharides. Bioinformatic analysis supports the conservation of the amylopectin-targeting platform in CBM74s from resistant-starch degrading bacteria. Our results suggest that RbCBM74 and RbCBM26 within Sas6 recognize discrete aspects of the starch granule, providing molecular insight into how this structure is accommodated by gut bacteria.

Breaking the radiation damage limit with Cryo-SAXS.

Small angle x-ray scattering (SAXS) is a versatile and widely used technique for obtaining low-resolution structures of macromolecules and complexes. SAXS experiments measure molecules in solution, without the need for labeling or crystallization. However, radiation damage currently limits the application of SAXS to molecules that can be produced in microgram quantities; for typical proteins, 10-20 µL of solution at 1 mg/mL is required to accumulate adequate signal before irreversible x-ray damage is observed. Here, we show that cryocooled proteins and nucleic acids can withstand doses at least two orders of magnitude larger than room temperature samples. We demonstrate accurate T = 100 K particle envelope reconstructions from sample volumes as small as 15 nL, a factor of 1000 smaller than in current practice. Cryo-SAXS will thus enable structure determination of difficult-to-express proteins and biologically important, highly radiation-sensitive proteins including light-activated switches and metalloenzymes.

Global radiation damage: temperature dependence, time dependence and how to outrun it.

A series of studies that provide a consistent and illuminating picture of global radiation damage to protein crystals, especially at temperatures above ∼200 K, are described. The radiation sensitivity shows a transition near 200 K, above which it appears to be limited by solvent-coupled diffusive processes. Consistent with this interpretation, a component of global damage proceeds on timescales of several minutes at 180 K, decreasing to seconds near room temperature. As a result, data collection times of order 1 s allow up to half of global damage to be outrun at 260 K. Much larger damage reductions near room temperature should be feasible using larger dose rates delivered using microfocused beams, enabling a significant expansion of structural studies of proteins under more nearly native conditions.

BioXTAS RAW 2: new developments for a free open-source program for small angle scattering data reduction and analysis.

BioXTAS RAW is a free, open-source program for reduction, analysis and modelling of biological small angle scattering data. Here, the new developments in RAW version 2 are described. These include: improved data reduction using pyFAI; updated automated Guinier fitting and Dmax finding algorithms; automated series (e.g. SEC-SAXS) buffer and sample region finding algorithms; linear and integral baseline correction for series; deconvolution of series data using REGALS; creation of electron density reconstructions via DENSS; a comparison window showing residuals, ratios, and statistical comparisons between profiles; and generation of PDF reports with summary plots and tables for all analysis. In addition, there is now a RAW API, which can be used without the GUI, providing full access to all of the functionality found in the GUI. In addition to these new capabilities, RAW has undergone significant technical updates, such as adding Python 3 compatibility, and has entirely new documentation available both online and in the program.

GRB2 dimerization mediated by SH2 domain-swapping is critical for T cell signaling and cytokine production.

GRB2 is an adaptor protein required for facilitating cytoplasmic signaling complexes from a wide array of binding partners. GRB2 has been reported to exist in either a monomeric or dimeric state in crystal and solution. GRB2 dimers are formed by the exchange of protein segments between domains, otherwise known as "domain-swapping". Swapping has been described between SH2 and C-terminal SH3 domains in the full-length structure of GRB2 (SH2/C-SH3 domain-swapped dimer), as well as between α-helixes in isolated GRB2 SH2 domains (SH2/SH2 domain-swapped dimer). Interestingly, SH2/SH2 domain-swapping has not been observed within the full-length protein, nor have the functional influences of this novel oligomeric conformation been explored. We herein generated a model of full-length GRB2 dimer with an SH2/SH2 domain-swapped conformation supported by in-line SEC-MALS-SAXS analyses. This conformation is consistent with the previously reported truncated GRB2 SH2/SH2 domain-swapped dimer but different from the previously reported, full-length SH2/C-terminal SH3 (C-SH3) domain-swapped dimer. Our model is also validated by several novel full-length GRB2 mutants that favor either a monomeric or a dimeric state through mutations within the SH2 domain that abrogate or promote SH2/SH2 domain-swapping. GRB2 knockdown and re-expression of selected monomeric and dimeric mutants in a T cell lymphoma cell line led to notable defects in clustering of the adaptor protein LAT and IL-2 release in response to TCR stimulation. These results mirrored similarly-impaired IL-2 release in GRB2-deficient cells. These studies show that a novel dimeric GRB2 conformation with domain-swapping between SH2 domains and monomer/dimer transitions are critical for GRB2 to facilitate early signaling complexes in human T cells.