Recurrent activating mutations of CD28 in peripheral T-cell lymphomas.

Peripheral T-cell lymphomas (PTCLs) comprise a heterogeneous group of mature T-cell neoplasms with a poor prognosis. Recently, mutations in TET2 and other epigenetic modifiers as well as RHOA have been identified in these diseases, particularly in angioimmunoblastic T-cell lymphoma (AITL). CD28 is the major co-stimulatory receptor in T cells which, upon binding ligand, induces sustained T-cell proliferation and cytokine production when combined with T-cell receptor stimulation. We have identified recurrent mutations in CD28 in PTCLs. Two residues-D124 and T195-were recurrently mutated in 11.3% of cases of AITL and in one case of PTCL, not otherwise specified (PTCL-NOS). Surface plasmon resonance analysis of mutations at these residues with predicted differential partner interactions showed increased affinity for ligand CD86 (residue D124) and increased affinity for intracellular adaptor proteins GRB2 and GADS/GRAP2 (residue T195). Molecular modeling studies on each of these mutations suggested how these mutants result in increased affinities. We found increased transcription of the CD28-responsive genes CD226 and TNFA in cells expressing the T195P mutant in response to CD3 and CD86 co-stimulation and increased downstream activation of NF-κB by both D124V and T195P mutants, suggesting a potential therapeutic target in CD28-mutated PTCLs.

Human RAD52 protein has extreme thermal stability.

The human RAD52 protein plays an important role in the earliest stages of chromosomal double-strand break repair via the homologous recombination pathway. Individual subunits of RAD52 associate into seven-membered rings. These rings can form higher order complexes. RAD52 binds to DNA breaks, and recent studies suggest that the higher order self-association of the rings promotes DNA end joining. Monomers of the RAD52(1--192) deletion mutant also associate into ring structures but do not form higher order complexes. The thermal stability of wild-type and mutant RAD52 was studied by differential scanning calorimetry. Three thermal transitions (labeled A, B, and C) were observed with melting temperatures of 38.8, 73.1, and 115.2 degrees C. The RAD52(1--192) mutant had only two thermal transitions at 47.6 and 100.9 degrees C (labeled B and C). Transitions were labeled such that transition C corresponds to complete unfolding of the protein. The effect of temperature and protein concentration on RAD52 self-association was analyzed by dynamic light scattering. From these data a four-state hypothetical model was developed to explain the thermal denaturation profile of wild-type RAD52. The three thermal transitions in this model were assigned as follows. Transition A was attributed to the disruption of higher order assemblies of RAD52 rings, transition B to the disruption of rings to individual subunits, and transition C to complete unfolding. The ring-shaped quaternary structure of RAD52 and the formation of higher ordered complexes of rings appear to contribute to the extreme stability of RAD52. Higher ordered complexes of rings are stable at physiological temperatures in vitro.

A test of macromolecular crystallization in microgravity: large well ordered insulin crystals.

Crystals of insulin grown in microgravity on Space Shuttle Mission STS-95 were extremely well ordered and unusually large (many >2 mm). The physical characteristics of six microgravity and six earth-grown crystals were examined by X-ray analysis employing superfine phi slicing and unfocused synchrotron radiation. This experimental setup allowed hundreds of reflections to be precisely examined from each crystal in a short period of time. The microgravity crystals were on average 34 times larger, had sevenfold lower mosaicity, had 54-fold higher reflection peak heights and diffracted to significantly higher resolution than their earth-grown counterparts. A single mosaic domain model could account for the observed reflection profiles in microgravity crystals, whereas data from earth crystals required a model with multiple mosaic domains. This statistically significant and unbiased characterization indicates that the microgravity environment was useful for the improvement of crystal growth and the resultant diffraction quality in insulin crystals and may be similarly useful for macromolecular crystals in general.

Human RAD52 exhibits two modes of self-association.

The human RAD52 protein plays an important role in the earliest stages of chromosomal double-strand break repair via the homologous recombination pathway. Individual subunits of RAD52 self-associate into rings that can then form higher order complexes. RAD52 binds to double-strand DNA ends, and recent studies suggest that the higher order self-association of the rings promotes DNA end-joining. Earlier studies defined the self-association domain of RAD52 to a unique region in the N-terminal half of the protein. Here we show that there are in fact two experimentally separable self-association domains in RAD52. The N-terminal self-association domain mediates the assembly of monomers into rings, and the previously unidentified domain in the C-terminal half of the protein mediates higher order self-association of the rings.

Dynamic light-scattering analysis of full-length human RPA14/32 dimer: purification, crystallization and self-association.

Replication protein A (RPA) is a single-stranded DNA-binding protein involved in all aspects of eukaryotic DNA metabolism. A soluble heterodimeric form of RPA is composed of 14 and 32 kDa subunits (RPA14/32). Dynamic light-scattering (DLS) analysis was used to improve the purification, stabilization and crystallization of RPA14/32. Increasing the concentration of reducing agent in the last stage of purification diminished the size of a secondary peak in the anion-exchange chromatograph and promoted a single species in solution. This resulted in decreased polydispersity in the purified protein and enhanced the crystallization time from 9-12 months to 6 d. With this homogeneous preparation, the reversible association of RPA14/32 into a dimer of dimers was demonstrated by DLS. Four different crystal forms of RPA14/32 were obtained for structure determination and complete diffraction data were collected using synchrotron radiation for three of them. Data to 2.4 A resolution was collected from hexagonal crystals (P3(2) or P3(1); a = b = 63.0, c = 272.6 A) and to 2.2 and 1.9 A resolution from two orthorhombic crystal forms (both P2(1)2(1)2(1); form I, a = 61.4, b = 75.2, c = 131.6 A; form II, a = 81.8, b = 140.4, c = 173.1 A).

The high-mosaicity illusion: revealing the true physical characteristics of macromolecular crystals.

Typical measurements of macromolecular crystal mosaicity are dominated by the characteristics of the X-ray beam and as a result the mosaicity value given during data processing can be an artifact of the instrumentation rather than the sample. For physical characterization of crystals, an experimental system and software have been developed to simultaneously measure the diffraction resolution and mosaic spread of macromolecular crystals. The contributions of the X-ray beam to the reflection angular widths were minimized by using a highly parallel, highly monochromatic synchrotron source. Hundreds of reflection profiles over a wide resolution range were rapidly measured using a charge-coupled device (CCD) area detector in combination with superfine phi-slicing data collection. The Lorentz effect and beam contributions were evaluated and deconvoluted from the recorded data. Data collection and processing is described. From 1 degrees of superfine phi-slice data collected on a crystal of manganese superoxide dismutase, the mosaicities of 260 reflections were measured. The average mosaicity was 0.0101 degrees (s.d. 0.0035 degrees ) measured as the full-width at half-maximum (FWHM) and ranged from 0.0011 to 0. 0188 degrees. Each reflection profile was individually fitted with two Gaussian profiles, with the first Gaussian contributing 55% (s.d. 9%) and the second contributing 35% (s.d. 9%) of the reflection. On average, the deconvoluted width of the first Gaussian was 0.0054 degrees (s.d. 0.0015 degrees ) and the second was 0.0061 degrees (s. d. 0.0023 degrees ). The mosaicity of the crystal was anisotropic, with FWHM values of 0.0068, 0.0140 and 0.0046 degrees along the a, b and c axes, respectively. The anisotropic mosaicity analysis indicates that the crystal is most perfect in the direction that corresponds to the favored growth direction of the crystal.

Cryo-trapping the six-coordinate, distorted-octahedral active site of manganese superoxide dismutase.

Superoxide dismutase protects organisms from potentially damaging oxygen radicals by catalyzing the disproportionation of superoxide to oxygen and hydrogen peroxide. We report the use of cryogenic temperatures to kinetically capture the sixth ligand bound to the active site of manganese superoxide dismutase (MnSOD). Synchrotron X-ray diffraction data was collected from Escherichia coli MnSOD crystals grown at pH 8.5 and cryocooled to 100 K. Structural refinement to 1.55 A resolution and close inspection of the active site revealed electron density for a sixth ligand that was interpreted to be a hydroxide ligand. The six-coordinate, distorted-octahedral geometry assumed during inhibition by hydroxide is compared to the room temperature, five-coordinate, trigonal bipyramidal active site determined with crystals grown from practically identical conditions. The gateway residues Tyr34, His30 and a tightly bound water molecule are implicated in closing-off the active site and blocking the escape route of the sixth ligand.

Ethylammonium nitrate: a protein crystallization reagent.

Ethylammonium nitrate (EAN) is a liquid organic salt that has many potential applications in protein chemistry. Because this solvent has hydrophobic and ionic character as well as the ability to hydrogen bond, it is especially well suited for broad use in protein crystallography. For example, EAN may be used as an additive, a detergent, a precipitating agent or to deliver ligands into protein crystals. A discussion of the crystallization of lysozyme using EAN as a precipitating agent is given here.

Crystal structure of Y34F mutant human mitochondrial manganese superoxide dismutase and the functional role of tyrosine 34.

Tyrosine 34 is a prominent and conserved residue in the active site of the manganese superoxide dismutases in organisms from bacteria to man. We have prepared the mutant containing the replacement Tyr 34 --> Phe (Y34F) in human manganese superoxide dismutase (hMnSOD) and crystallized it in two different crystal forms, orthorhombic and hexagonal. Crystal structures of hMnSOD Y34F have been solved to 1.9 A resolution in a hexagonal crystal form, denoted as Y34Fhex, and to 2.2 A resolution in an orthorhombic crystal form, denoted as Y34Fortho. Both crystal forms give structures that are closely superimposable with that of wild-type hMnSOD, with the phenyl rings of Tyr 34 in the wild type and Phe 34 in the mutant very similar in orientation. Therefore, in Y34F, a hydrogen-bonded relay that links the metal-bound hydroxyl to ordered solvent (Mn-OH to Gln 143 to Tyr 34 to H2O to His 30) is broken. Surprisingly, the loss of the Tyr 34 hydrogen bonds resulted in large increases in stability (measured by Tm), suggesting that the Tyr 34 hydroxyl does not play a role in stabilizing active-site architecture. The functional role of the side chain hydroxyl of Tyr 34 can be evaluated by comparison of the Y34F mutant with the wild-type hMnSOD. Both wild-type and Y34F had kcat/Km near 10(9) M-1 s-1, close to diffusion-controlled; however, Y34F showed kcat for maximal catalysis smaller by 10-fold than the wild type. In addition, the mutant Y34F was more susceptible to product inhibition by peroxide than the wild-type enzyme. This activity profile and the breaking of the hydrogen-bonding chain at the active site caused by the replacement Tyr 34 --> Phe suggest that Tyr 34 is a proton donor for O2* - reduction to H2O2 or is involved indirectly by orienting solvent or other residues for proton transfer. Up to 100 mM buffers in solution failed to enhance catalysis by either Y34F or the wild-type hMnSOD, suggesting that protonation from solution cannot enhance the release of the inhibiting bound peroxide ion, likely reflecting the enclosure of the active site by conserved residues as shown by the X-ray structures. The increased thermostability of the mutant Y34F and equal diffusion-controlled activity of Y34F and wild-type enzymes with normal superoxide levels suggest that evolutionary conservation of active-site residues in metalloenzymes reflects constraints from extreme rather than average cellular conditions. This new hypothesis that extreme rather than normal substrate concentrations are a powerful constraint on residue conservation may apply most strongly to enzyme defenses where the ability to meet extreme conditions directly affects cell survival.

Structure of a protein photocycle intermediate by millisecond time-resolved crystallography.

The blue-light photoreceptor photoactive yellow protein (PYP) undergoes a self-contained light cycle. The atomic structure of the bleached signaling intermediate in the light cycle of PYP was determined by millisecond time-resolved, multiwavelength Laue crystallography and simultaneous optical spectroscopy. Light-induced trans-to-cis isomerization of the 4-hydroxycinnamyl chromophore and coupled protein rearrangements produce a new set of active-site hydrogen bonds. An arginine gateway opens, allowing solvent exposure and protonation of the chromophore's phenolic oxygen. Resulting changes in shape, hydrogen bonding, and electrostatic potential at the protein surface form a likely basis for signal transduction. The structural results suggest a general framework for the interpretation of protein photocycles.

Human mitochondrial manganese superoxide dismutase polymorphic variant Ile58Thr reduces activity by destabilizing the tetrameric interface.

Human manganese superoxide dismutase (MnSOD) is a homotetrameric enzyme which protects mitochondria against oxygen-mediated free radical damage. Within each subunit, both the N-terminal helical hairpin and C-terminal alpha/beta domains contribute ligands to the catalytic manganese site. Two identical four-helix bundles, symmetrically assembled from the N-terminal helical hairpins, form a novel tetrameric interface that stabilizes the active sites. The 2.5 A crystallographic structure of the naturally occurring polymorphic variant Ile58Thr MnSOD reveals that the helical hairpin mutation Thr58 causes two packing defects in each of the two four-helix bundles of the tetrameric interface. Similar mutations, expected to cause packing defects in the Cu,ZnSOD dimer interface, are associated with the degenerative disease amyotrophic lateral sclerosis. Ile58Thr MnSOD is primarily dimeric in solution and is significantly less thermostable than the normal enzyme, with decreases of 15 degrees C in the main melting temperature and 20 degrees C in the heat-inactivation temperature. Consequently, this mutant MnSOD is compromised at normal body temperatures: thermal inactivation, predicted from the decrease in thermal stability, occurs with a theoretical half-life of only 3.2 h at 37 degrees C (1.4 h at 41 degrees C), compared with 3.1 years for native MnSOD. This prediction is supported by direct measurements: incubation at 41.7 degrees C for 3 h has no effect on the activity of native MnSOD but completely inactivates mutant MnSOD. Rapid inactivation of Ile58Thr MnSOD at the elevated temperatures associated with fever and inflammation could provide an early advantage by killing infected cells, but also would increase superoxide-mediated oxidative damage and perhaps contribute to late-onset diseases.

1.4 A structure of photoactive yellow protein, a cytosolic photoreceptor: unusual fold, active site, and chromophore.

A photosensing protein directs light energy captured by its chromophore into a photocycle. The protein's structure must accommodate the photocycle and promote the resulting chemical or conformational changes that lead to signal transduction. The 1.4 A crystallographic structure of photoactive yellow protein, determined by multiple isomorphous replacement methods, provides the first view at atomic resolution of a protein with a photocycle. The alpha/beta fold, which differs from the original chain tracing, shows striking similarity to distinct parts of the signal transduction proteins profilin and the SH2 domain. In the dark state structure of photoactive yellow protein, the novel 4-hydroxycinnamyl chromophore, covalently attached to Cys69, is buried within the major hydrophobic core of the protein and is tethered at both ends by hydrogen bonds. In the active site, the yellow anionic form of the chromophore is stabilized by hydrogen bonds from the side chains of Tyr42 and buried Glu46 to the phenolic oxygen atom and by electrostatic complementarity with the positively charged guanidinium group of Arg52. Thr50 further interlocks Tyr42, Glu46, and Arg52 through a network of active site hydrogen bonds. Arg52, located in a concavity of the protein surface adjacent to the dominant patch of negative electrostatic potential, shields the chromophore from solvent and is positioned to form a gateway for the phototactic signal. Overall, the high-resolution structure of photoactive yellow protein supports a mechanism whereby electrostatic interactions create an active site poised for photon-induced rearrangements and efficient protein-mediated signal transduction.

Complete chemical structure of photoactive yellow protein: novel thioester-linked 4-hydroxycinnamyl chromophore and photocycle chemistry.

The unique ability of photoactive proteins to capture and use energy from a photon of light depends on the chromophore, its linkage to the protein, and the surrounding protein environment. To understand the molecular mechanisms by which a chromophore and protein interact to undergo a light cycle, we are studying photoactive yellow protein (PYP), a 14-kDa water-soluble photoreceptor from Ectothiorhodospira halophila with a photocycle similar to that of sensory rhodopsin. Here, we report the cloning and sequencing of the pyp gene and the chemical identification of both the chromophore and its covalent linkage to the protein. Elemental composition data from high-resolution mass spectrometry of a proteolytically derived chromopeptide, pH titrations and UV-visible spectroscopy of the protein-bound and chemically released chromophore, and fragmentation mass spectrometry of the liberated chromophore amide were combined with results from the 1.4-A-resolution protein crystal structure to identify the chromophore in PYP as a 4-hydroxycinnamyl group covalently bound to the sole cysteine residue via a thioester linkage. While 4-hydroxycinnamate is a metabolic product of the phenylpropanoid pathway and a key molecule in plant stress response, this is the first report of covalent modification of a protein by this group. In the dark (yellow) state of PYP, the protein stabilizes the chromophore as the deprotonated phenolate anion. By combining our biochemical characterization of the chromophore with other published observations, we propose a chemical basis for the photocycle: following the initial absorption of a photon, the photocycle of PYP involves protonation of the chromophore to a neutral phenol form corresponding to the observed photobleached intermediate.

The 1.8 A structure of carbonmonoxy-beta 4 hemoglobin. Analysis of a homotetramer with the R quaternary structure of liganded alpha 2 beta 2 hemoglobin.

The beta-chains isolated from the human hemoglobin alpha 2 beta 2 heterotetramer self-assemble to form a beta 4 homotetramer. We report the structure of the carbonmonoxy-beta 4 (CO beta 4) tetramer refined at a resolution of 1.8 A. Compared to the three known quaternary structures of human hemoglobin, the T state, the R state and the R2 state, the quaternary structure of CO beta 4 most closely resembles the R state. While the degree of structural similarity between CO beta 4 and the R state of liganded alpha 2 beta 2 is quite high, differences between the alpha and beta-chain sequences result in interesting alternative packing arrangements at the subunit interfaces of CO beta 4. In particular, Arg40 beta and Asp99 beta interact across the CO beta 4 equivalent of the alpha 1 beta 2 interface to form two symmetry-related salt bridges that have no counterpart in either liganded or deoxyhemoglobin. Because these salt bridges are near a 2-fold symmetry axis, steric constraints prevent their simultaneous formation, and electron density images of Arg40 beta and Asp99 beta show equally populated dual conformations for the side-chains of both residues. Relative to the liganded alpha 2 beta 2 tetramer, the Arg40 beta...Asp99 beta salt bridges introduce ionic interactions that should strengthen the CO beta 4 tetramer. The CO beta 4 equivalent of the alpha 1 alpha 2 and beta 1 beta 2 interfaces strengthens the tetramer relative to the liganded alpha 2 beta 2 tetramer by tethering both ends of the central cavity. (The entrance to the central cavity is altered so that the N termini move closer together and the C termini further apart, forming an anion binding pocket that is absent in liganded alpha 2 beta 2 hemoglobin.) In contrast, analysis of the CO beta 4 counterpart of the alpha 1 beta 1 interface indicates that this interface is weakened in the CO beta 4 tetramer. These differences in interface stability provide a structural explanation for the published observation that the alpha 2 beta 2 tetramer assembles via a stable alpha 1 beta 1 dimer intermediate, whereas assembly of the CO beta 4 tetramer is characterized more accurately by a monomer-tetramer equilibrium.

The 1.9 A structure of deoxy beta 4 hemoglobin. Analysis of the partitioning of quaternary-associated and ligand-induced changes in tertiary structure.

The crystal structure of the deoxygenated form of the human hemoglobin beta 4 tetramer (deoxy beta 4) has been determined and refined at a resolution of 1.9 A. A detailed comparison of the quaternary structures of carbonmonoxy-beta 4 (CO beta 4) and deoxy beta 4 shows that ligand binding to the beta 4 tetramer produces only slight movements of the subunits relative to each other. Therefore, unlike the hemoglobin alpha 2 beta 2 tetramer, where the transition from an unliganded T state tetramer to a liganded R state tetramer results in a large change in quaternary structure, beta 4 is locked in a quaternary structure that very closely resembles the R state. By comparing the high-resolution structures of T state deoxy alpha 2 beta 2, R state deoxy beta 4 and R state CO beta 4, it is possible to partition the changes in beta subunit tertiary structure into those that arise from changes in quaternary structure and those that result solely from ligand binding. Specifically, when viewed from the heme reference frame, comparison of the structures of T state deoxy alpha 2 beta 2 and R state deoxy beta 4 shows that the T-to-R quaternary structure transition induces changes in beta subunit tertiary structure that are approximately halfway toward the tertiary structure observed in liganded beta 4 and liganded alpha 2 beta 2. When viewed from the reference frame of the globin backbone atoms, the T-to-R quaternary structure transition induces a small rotation of the heme group and a shift of the "allosteric core" (the end of the F helix, the FG corner, the beginning of the G helix, and the heme group) away from the E helix. These movements open the ligand binding pocket and place the heme in a more symmetric position relative to the proximal histidine residue. Together, these effects work in unison to give the subunits of deoxy beta 4 a tertiary structure that has high ligand affinity.

The structure of human mitochondrial manganese superoxide dismutase reveals a novel tetrameric interface of two 4-helix bundles.

The 2.2 A resolution crystal structure of recombinant human manganese superoxide dismutase, a homotetrameric enzyme that protects mitochondria against oxygen-mediated free radical damage, has been determined. Within each subunit, both the N-terminal helical hairpin and C-terminal alpha/beta domains contribute ligands to the catalytic manganese site. Two identical 4-helix bundles, symmetrically assembled from the N-terminal helical hairpins, form novel tetrameric interfaces that stabilize the active sites. Structurally altered polymorphic variants with reduced activity, such as tetrameric interface mutant Ile-58 to Thr, may produce not only an early selective advantage, through enhanced cytotoxicity of tumor necrosis factor for virus-infected cells, but also detrimental effects from increased mitochondrial oxidative damage, contributing to degenerative conditions, including diabetes, aging, and Parkinson's and Alzheimer's diseases.