Sergei Radaev

CONFORMATIONAL PLASTICITY REVEALED BY THE COCRYSTAL STRUCTURE OF NKG2D AND ITS CLASS I MHC-LIKE LIGAND ULBP3

NKG2D is known to trigger the natural killer (NK) cell lysis of various tumor and virally infected cells. In the NKG2D/ULBP3 complex, the structure of ULBP3 resembles the alpha1 and alpha2 domains of classical MHC molecules without a bound peptide. The lack of alpha3 and beta2m domains is compensated by replacing two hydrophobic patches at the underside of the class I MHC-like beta sheet floor with a group of hydrophilic and charged residues in ULBP3. NKG2D binds diagonally across the ULBP3 alpha helices, creating a complementary interface, an asymmetrical subunit orientation, and local conformational adjustments in the receptor. The interface is stabilized primarily by hydrogen bonds and hydrophobic interactions. Unlike the KIR receptors that recognize a conserved HLA region by a lock-and-key mechanism, NKG2D recognizes diverse ligands by an induced-fit mechanism.

Crystal Structure of the Extracellular Domain of a Human FcγRIII

Fc receptors play a major role in immune defenses against pathogens and in inflammatory processes. The crystal structure of a human immunoglobulin receptor, FcgammaRIIIb, has been determined to 1.8 A resolution. The overall fold consists of two immunoglobulin-like domains with an acute interdomain hinge angle of approximately 50 degrees. Trp-113, wedged between the N-terminal D1 and the C-terminal D2 domains, appears to further restrict the hinge angle. The putative Fc binding region of the receptor carries a net positive charge complementary to the negative-charged receptor binding regions on Fc. A 1:1 binding stoichiometry between the receptor and Fc was measured by both the equilibrium and nonequilibrium size-exclusion chromatography. Two separate parallel dimers are observed in the crystal lattice, offering intriguing models for receptor aggregation.

Ternary Complex of Transforming Growth Factor-β1 Reveals Isoform-specific Ligand Recognition and Receptor Recruitment in the Superfamily

Transforming growth factor (TGF)-β1, -β2, and -β3 are 25-kDa homodimeric polypeptides that play crucial nonoverlapping roles in embryogenesis, tissue development, carcinogenesis, and immune regulation. Here we report the 3.0-Å resolution crystal structure of the ternary complex between human TGF-β1 and the extracellular domains of its type I and type II receptors, TβRI and TβRII. The TGF-β1 ternary complex structure is similar to previously reported TGF-β3 complex except with a 10° rotation in TβRI docking orientation. Quantitative binding studies showed distinct kinetics between the receptors and the isoforms of TGF-β. TβRI showed significant binding to TGF-β2 and TGF-β3 but not TGF-β1, and the binding to all three isoforms of TGF-β was enhanced considerably in the presence of TβRII. The preference of TGF-β2 to TβRI suggests a variation in its receptor recruitment in vivo. Although TGF-β1 and TGF-β3 bind and assemble their ternary complexes in a similar manner, their structural differences together with differences in the affinities and kinetics of their receptor binding may underlie their unique biological activities. Structural comparisons revealed that the receptor-ligand pairing in the TGF-β superfamily is dictated by unique insertions, deletions, and disulfide bonds rather than amino acid conservation at the interface. The binding mode of TβRII on TGF-β is unique to TGF-βs, whereas that of type II receptor for bone morphogenetic protein on bone morphogenetic protein appears common to all other cytokines in the superfamily. Further, extensive hydrogen bonds and salt bridges are present at the high affinity cytokine-receptor interfaces, whereas hydrophobic interactions dominate the low affinity receptor-ligand interfaces.

The 1.1 Å Crystal Structure of Human TGF-β Type II Receptor Ligand Binding Domain

Transforming growth factor beta (TGF-beta) is involved in a wide range of biological functions including development, carcinogenesis, and immune regulation. Here we report the 1.1 A resolution crystal structure of human TGF-beta type II receptor ectodomain (TBRII). The overall structure of TBRII is similar to that of activin type II receptor ectodomain (ActRII) and bone morphogenic protein receptor type IA (BRIA). It displays a three-finger toxin fold with fingers formed by the beta strand pairs beta1-beta2, beta3-beta4, and beta5-beta6. The first finger in the TBRII is significantly longer than in ActRII and BRIA and folds tightly between the second finger and the C terminus. Surface charge distributions and hydrophobic patches predict potential TBRII binding sites.

Making Sense of the Diverse Ligand Recognition by NKG2D

NKG2D recognizes multiple diverse ligands. Despite recent efforts in determining the crystal structures of NKG2D-ligand complexes, the principle governing this receptor-ligand recognition and hence the criteria for identifying unknown ligands of NKG2D remain central issues to be resolved. Here we compared the molecular recognition between NKG2D and three of the known ligands, UL16 binding protein (ULBP), MHC class I-like molecule, and retinoic acid early inducible gene as observed in the ligand-complexed crystal structures. The comparison shows that while the receptor uses a common interface region to bind the three diverse ligands, each ligand forms a distinct, but overlapping, set of hydrogen bonds, hydrophobic interactions, and salt bridges, illustrating the underlying principle of NKG2D-ligand recognition being the conservation in overall shape complementarity and binding energy while permitting variation in ligand sequence through induced fit recognition. To further test this hypothesis and to distinguish between diverse recognition and promiscuous ligand binding, four ULBP3 interface mutations, H21A, E76A, R82M, and D169A, were generated to each disrupt a single hydrogen bond or salt bridge. All mutant ULBP3 displayed reduced receptor binding, suggesting a specific, rather than promiscuous, receptor-ligand recognition. Mutants with severe loss of binding affect the receptor interactions that are mostly buried. Finally, a receptor-ligand recognition algorithm was developed to assist the identification of diverse NKG2D ligands based on evaluating the potential hydrogen bonds, hydrophobic interactions, and salt bridges at the receptor-ligand interface.

A survey of protein-protein complex crystallizations.

A survey of crystallization conditions was carried out for 650 published protein-protein complexes in the Protein Data Bank (PDB) of the Research Collaboratory for Structural Bioinformatics (RCSB). This resulted in the establishment of a Protein Complex Crystallization Database (PCCD) and a set of configuration-space boundaries for protein-complex crystallizations. Overall, polyethylene glycol (PEG) based conditions accounted for 70-80% of all crystallizations, with PEG 3000-4000, 5000-6000 and 8000 being the most frequently used. The median values of PEG concentrations were between 10 and 20% and were inversely correlated with their molecular weights. Ammonium sulfate remained the most favorable salt precipitant, with a median concentration of 1.6 M. The crystallization pH for the vast majority of protein complexes was between 5.0 and 8.0. Overall, the boundaries for the crystallization configuration space of protein complexes appear to be more restricted than those of soluble proteins. This may reflect the limited stability and solubility of protein-protein complexes. Based on statistical analysis of the database, a sparse-matrix and a systematic buffer and pH screen were formulated to best represent the crystallization of protein complexes.

Structural and Functional Studies of Igαβ and Its Assembly with the B Cell Antigen Receptor

The B cell antigen receptor (BCR) plays an essential role in all phases of B cell development. Here we show that the extracellular domains of murine and human Igbeta form an I-set immunoglobulin-like structure with an interchain disulfide between cysteines on their G strands. Structural and sequence analysis suggests that Igalpha displays a similar fold as Igbeta. An Igalphabeta heterodimer model was generated based on the unique disulfide-bonded Igbeta dimer. Solution binding studies showed that the extracellular domains of Igalphabeta preferentially recognize the constant region of BCR with mu chain specificity, suggesting a role for Igalphabeta to enhance BCRmu chain signaling. Cluster mutations on Igalpha, Igbeta, and a membrane-bound form of immunoglobulin (mIgM) based on the structural model identified distinct areas of potential contacts involving charged residues on both subunits of the coreceptor and the Cmu4 domain of mIgM. These studies provide the first structural model for understanding BCR function.

Free IL-12p40 Monomer Is a Polyfunctional Adaptor for Generating Novel IL-12–like Heterodimers Extracellularly

IL-12p40 partners with the p35 and p19 polypeptides to generate the heterodimeric cytokines IL-12 and IL-23, respectively. These cytokines play critical and distinct roles in host defense. The assembly of these heterodimers is thought to take place within the cell, resulting in the secretion of fully functional cytokines. Although the p40 subunit alone can also be rapidly secreted in response to inflammatory signals, its biological significance remains unclear. In this article, we show that the secreted p40 monomer can generate de novo IL-12–like activities by combining extracellularly with p35 released from other cells. Surprisingly, an unbiased proteomic analysis reveals multiple such extracellular binding partners for p40 in the serum of mice after an endotoxin challenge. We biochemically validate the binding of one of these novel partners, the CD5 Ag-like glycoprotein, to the p40 monomer. Nevertheless, the assembled p40-CD5L heterodimer does not recapitulate the biological activity of IL-12. These findings underscore the plasticity of secreted free p40 monomer, suggesting that p40 functions as an adaptor that is able to generate multiple de novo composites in combination with other locally available polypeptide partners after secretion.

Crystallization of protein-protein complexes

Crystallizing protein–protein complexes remains a rate-limiting step in their structure characterization. Crystallization conditions for the known protein–protein complexes have been surveyed in both the Protein Data Bank and the BMCD database. Compared with non-complexed proteins, crystallization conditions for protein–protein complexes are less diverse and heavily favor (71% versus 27%) polyethylene glycols (PEG) rather than ammonium sulfate or other high-salt crystallization conditions. The results suggest that the stability of protein complexes limits their available crystallization configuration space. Based on the survey, a set of sparse-matrix screen conditions was designed.

A rational approach to heavy-atom derivative screening

In order to overcome the difficulties associated with the ‘classical’ heavy-atom derivatization procedure, an attempt has been made to develop a rational crystal-free heavy-atom-derivative screening method and a quick-soak derivatization procedure which allows heavy-atom compound identification.