Claudine Darnault

Ni-Zn-[Fe 4 -S 4 ] and Ni-Ni-[Fe 4 -S 4 ] clusters in closed and open α subunits of acetyl-CoA synthase/carbon monoxide dehydrogenase

The crystal structure of the tetrameric α2β2 acetyl-coenzyme A synthase/carbon monoxide dehydrogenase from Moorella thermoacetica has been solved at 1.9 Å resolution. Surprisingly, the two α subunits display different (open and closed) conformations. Furthermore, X-ray data collected from crystals near the absorption edges of several metal ions indicate that the closed form contains one Zn and one Ni at its active site metal cluster (A-cluster) in the α subunit, whereas the open form has two Ni ions at the corresponding positions. Alternative metal contents at the active site have been observed in a recent structure of the same protein in which A-clusters contained one Cu and one Ni, and in reconstitution studies of a recombinant apo form of a related acetyl-CoA synthase. On the basis of our observations along with previously reported data, we postulate that only the A-clusters containing two Ni ions are catalytically active.

CDR3 loop flexibility contributes to the degeneracy of TCR recognition.

T cell receptor (TCR) binding degeneracy lies at the heart of several physiological and pathological phenomena, yet its structural basis is poorly understood. We determined the crystal structure of a complex involving the BM3.3 TCR and an octapeptide (VSV8) bound to the H-2Kb major histocompatibility complex molecule at a 2.7 Å resolution, and compared it with the BM3.3 TCR bound to the H-2Kb molecule loaded with a peptide that has no primary sequence identity with VSV8. Comparison of these structures showed that the BM3.3 TCR complementarity-determining region (CDR) 3α could undergo rearrangements to adapt to structurally different peptide residues. Therefore, CDR3 loop flexibility helps explain TCR binding cross-reactivity.

Crystal structure of a T cell receptor bound to an allogeneic MHC molecule.

Many T cell receptors (TCRs) that are selected to respond to foreign peptide antigens bound to self major histocompatibility complex (MHC) molecules are also reactive with allelic variants of self-MHC molecules. This property, termed alloreactivity, causes graft rejection and graft-versus-host disease. The structural features of alloreactivity have yet to be defined. We now present a basis for this cross-reactivity, elucidated by the crystal structure of a complex involving the BM3.3 TCR and a naturally processed octapeptide bound to the H-2Kb allogeneic MHC class I molecule. A distinguishing feature of this complex is that the eleven-residue-long complementarity-determining region 3 (CDR3) found in the BM3.3 TCRα chain folds away from the peptide binding groove and makes no contact with the bound peptide, the latter being exclusively contacted by the BM3.3 CDR3β. Our results formally establish that peptide-specific, alloreactive TCRs interact with allo-MHC in a register similar to the one they use to contact self-MHC molecules.

A T Cell Receptor CDR3β Loop Undergoes Conformational Changes of Unprecedented Magnitude Upon Binding to a Peptide/MHC Class I Complex

The elongated complementary-determining region (CDR) 3beta found in the unliganded KB5-C20 TCR protrudes from the antigen binding site and prevents its docking onto the peptide/MHC (pMHC) surface according to a canonical diagonal orientation. We now present the crystal structure of a complex involving the KB5-C20 TCR and an octapeptide bound to the allogeneic H-2K(b) MHC class I molecule. This structure reveals how a tremendously large CDR3beta conformational change allows the KB5-C20 TCR to adapt to the rather constrained pMHC surface and achieve a diagonal docking mode. This extreme case of induced fit also shows that TCR plasticity is primarily restricted to CDR3 loops and does not propagate away from the antigen binding site.

C1Q Binds Phosphatidylserine and Likely Acts as a Multiligand-Bridging Molecule in Apoptotic Cell Recognition.

Efficient apoptotic cell clearance is critical for maintenance of tissue homeostasis, and to control the immune responses mediated by phagocytes. Little is known about the molecules that contribute “eat me” signals on the apoptotic cell surface. C1q, the recognition unit of the C1 complex of complement, also senses altered structures from self and is a major actor of immune tolerance. HeLa cells were rendered apoptotic by UV-B treatment and a variety of cellular and molecular approaches were used to investigate the nature of the target(s) recognized by C1q. Using surface plasmon resonance, C1q binding was shown to occur at early stages of apoptosis and to involve recognition of a cell membrane component. C1q binding and phosphatidylserine (PS) exposure, as measured by annexin V labeling, proceeded concomitantly, and annexin V inhibited C1q binding in a dose-dependent manner. As shown by cosedimentation, surface plasmon resonance, and x-ray crystallographic analyses, C1q recognized PS specifically and avidly (KD = 3.7–7 × 10−8 M), through multiple interactions between its globular domain and the phosphoserine group of PS. Confocal microscopy revealed that the majority of the C1q molecules were distributed in membrane patches where they colocalized with PS. In summary, PS is one of the C1q ligands on apoptotic cells, and C1q-PS interaction takes place at early stages of apoptosis, in newly organized membrane patches. Given its versatile recognition properties, these data suggest that C1q has the unique ability to sense different markers which collectively would provide strong eat me signals, thereby allowing efficient apoptotic cell removal.

X-ray crystallographic and computational studies of the O2-tolerant [NiFe]-hydrogenase 1 from Escherichia coli

The crystal structure of the membrane-bound O2-tolerant [NiFe]-hydrogenase 1 from Escherichia coli (EcHyd-1) has been solved in three different states: as-isolated, H2-reduced, and chemically oxidized. As very recently reported for similar enzymes from Ralstonia eutropha and Hydrogenovibrio marinus, two supernumerary Cys residues coordinate the proximal [FeS] cluster in EcHyd-1, which lacks one of the inorganic sulfide ligands. We find that the as-isolated, aerobically purified species contains a mixture of at least two conformations for one of the cluster iron ions and Glu76. In one of them, Glu76 and the iron occupy positions that are similar to those found in O2-sensitive [NiFe]-hydrogenases. In the other conformation, this iron binds, besides three sulfur ligands, the amide N from Cys20 and one Oϵ of Glu76. Our calculations show that oxidation of this unique iron generates the high-potential form of the proximal cluster. The structural rearrangement caused by oxidation is confirmed by our H2-reduced and oxidized EcHyd-1 structures. Thus, thanks to the peculiar coordination of the unique iron, the proximal cluster can contribute two successive electrons to secure complete reduction of O2 to H2O at the active site. The two observed conformations of Glu76 are consistent with this residue playing the role of a base to deprotonate the amide moiety of Cys20 upon iron binding and transfer the resulting proton away, thus allowing the second oxidation to be electroneutral. The comparison of our structures also shows the existence of a dynamic chain of water molecules, resulting from O2 reduction, located near the active site.

Low-temperature switching by photoinduced protonation in photochromic fluorescent proteins.

We have studied the photoswitching behaviour of a number of photochromic fluorescent proteins at cryo-temperature. Spectroscopic investigations at the ensemble level showed that EYFP, Dronpa and IrisFP all exhibit reversible photoswitching at 100 K, albeit with a low quantum yield. The photophysics of the process were studied in more details in the case of EYFP. The data suggest that photoinduced protonation of the chromophore is responsible for off-switching at cryo-temperature, and thus is possible in the absence of significant conformational freedom. This finding is consistent with the hypothesis that chromophore protonation may precede large amplitude conformational changes such as cis-trans isomerisation during off-photoswitching at room temperature. However, our data suggest that low-barrier photoinduced protonation pathways may in fact compete with room-temperature off-switching reactions in photochromic fluorescent proteins. The occurrence of reversible photoswitching at low-temperature is of interest to envisage cryo-nanoscopy experiments using genetically encoded fluorophores.

Simultaneous Measurements of Solvent Dynamics and Functional Kinetics in a Light-Activated Enzyme

Solvent fluctuations play a key role in controlling protein motions and biological function. Here, we have studied how individual steps of the reaction catalyzed by the light-activated enzyme protochlorophyllide oxidoreductase (POR) couple with solvent dynamics. To simultaneously monitor the catalytic cycle of the enzyme and the dynamical behavior of the solvent, we designed temperature-dependent UV-visible microspectrophotometry experiments, using flash-cooled nanodroplets of POR to which an exogenous soluble fluorophore was added. The formation and decay of the first two intermediates in the POR-catalyzed reaction were measured, together with the solvent glass transition and the buildup of crystalline ice at cryogenic temperatures. We find that formation of the first intermediate occurs below the glass transition temperature (T(g)), and is not affected by changes in solvent dynamics induced by modifying the glycerol content. In contrast, formation of the second intermediate occurs above T(g) and is influenced by changes in glycerol concentration in a manner remarkably similar to the buildup of crystalline ice. These results suggest that internal, nonslaved protein motions drive the first step of the POR-catalyzed reaction whereas solvent-slaved motions control the second step. We propose that the concept of solvent slaving applies to complex enzymes such as POR.

The Crystal Structure of Fe4S4 Quinolinate Synthase Unravels an Enzymatic Dehydration Mechanism That Uses Tyrosine and a Hydrolase-Type Triad

Quinolinate synthase (NadA) is a Fe4S4 cluster-containing dehydrating enzyme involved in the synthesis of quinolinic acid (QA), the universal precursor of the essential nicotinamide adenine dinucleotide (NAD) coenzyme. A previously determined apo NadA crystal structure revealed the binding of one substrate analog, providing partial mechanistic information. Here, we report on the holo X-ray structure of NadA. The presence of the Fe4S4 cluster generates an internal tunnel and a cavity in which we have docked the last precursor to be dehydrated to form QA. We find that the only suitably placed residue to initiate this process is the conserved Tyr21. Furthermore, Tyr21 is close to a conserved Thr-His-Glu triad reminiscent of those found in proteases and other hydrolases. Our mutagenesis data show that all of these residues are essential for activity and strongly suggest that Tyr21 deprotonation, to form the reactive nucleophilic phenoxide anion, is mediated by the triad. NadA displays a dehydration mechanism significantly different from the one found in archetypical dehydratases such as aconitase, which use a serine residue deprotonated by an oxyanion hole. The X-ray structure of NadA will help us unveil its catalytic mechanism, the last step in the understanding of NAD biosynthesis.

The crystal structure of the global anaerobic transcriptional regulator FNR explains its extremely fine-tuned monomer-dimer equilibrium.

The dimerization of the O2 sensor FNR is regulated by extremely fine-tuned interactions. The structure of the dimeric holo–fumarate and nitrate reduction regulator (FNR) from Aliivibrio fischeri has been solved at 2.65 Å resolution. FNR globally controls the transition between anaerobic and aerobic respiration in facultative anaerobes through the assembly/degradation of its oxygen-sensitive [4Fe-4S] cluster. In the absence of O2, FNR forms a dimer and specifically binds to DNA, whereas in its presence, the cluster is degraded causing FNR monomerization and DNA dissociation. We have used our crystal structure and the information previously gathered from numerous FNR variants to propose that this process is governed by extremely fine-tuned interactions, mediated by two salt bridges near the amino-terminal cluster-binding domain and an “imperfect” coiled-coil dimer interface. [4Fe-4S] to [2Fe-2S] cluster degradation propagates a conformational signal that indirectly causes monomerization by disrupting the first of these interactions and unleashing the “unzipping” of the FNR dimer in the direction of the carboxyl-terminal DNA binding domain.