Przemyslaw Grudnik

Protein targeting by the signal recognition particle

Abstract Protein targeting by the signal recognition particle (SRP) is universally conserved and starts with the recognition of a signal sequence in the context of a translating ribosome. SRP54 and FtsY, two multidomain proteins with guanosine triphosphatase (GTPase) activity, are the central elements of the SRP system. They have to coordinate the presence of a signal sequence with the presence of a vacant translocation channel in the membrane. For coordination the two GTPases form a unique, nearly symmetric heterodimeric complex in which the activation of GTP hydrolysis plays a key role for membrane insertion of substrate proteins. Recent results are integrated in an updated perception of the order of events in SRP-mediated protein targeting.

T cell Activation Is Driven by an ADP-Dependent Glucokinase Linking Enhanced Glycolysis with Mitochondrial Reactive Oxygen Species Generation

Mitochondria-originating reactive oxygen species (ROS) control T cell receptor (TCR)-induced gene expression. Here, we show that TCR-triggered activation of ADP-dependent glucokinase (ADPGK), an alternative, glycolytic enzyme typical for Archaea, mediates generation of the oxidative signal. We also show that ADPGK is localized in the endoplasmic reticulum and suggest that its active site protrudes toward the cytosol. The ADPGK-driven increase in glycolytic metabolism coincides with TCR-induced glucose uptake, downregulation of mitochondrial respiration, and deviation of glycolysis toward mitochondrial glycerol-3-phosphate dehydrogenase(GPD) shuttle; i.e., a metabolic shift to aerobic glycolysis similar to the Warburg effect. The activation of respiratory-chain-associated GPD2 results in hyperreduction of ubiquinone and ROS release from mitochondria. In parallel, mitochondrial bioenergetics and ultrastructure are altered. Downregulation of ADPGK or GPD2 abundance inhibits oxidative signal generation and induction of NF-κB-dependent gene expression, whereas overexpression of ADPGK potentiates them.

Structural basis for small molecule targeting of the programmed death ligand 1 (PD-L1).

Targeting the PD-1/PD-L1 immunologic checkpoint with monoclonal antibodies has provided unprecedented results in cancer treatment in the recent years. Development of chemical inhibitors for this pathway lags the antibody development because of insufficient structural information. The first nonpeptidic chemical inhibitors that target the PD-1/PD-L1 interaction have only been recently disclosed by Bristol-Myers Squibb. Here, we show that these small-molecule compounds bind directly to PD-L1 and that they potently block PD-1 binding. Structural studies reveal a dimeric protein complex with a single small molecule which stabilizes the dimer thus occluding the PD-1 interaction surface of PD-L1s. The small-molecule interaction “hot spots” on PD-L1 surfaces suggest approaches for the PD-1/PD-L1 antagonist drug discovery.

Insights into eukaryotic Rubisco assembly - Crystal structures of RbcX chaperones from Arabidopsis thaliana.

BACKGROUND Chloroplasts were formed by uptake of cyanobacteria into eukaryotic cells ca. 1.6 billion years ago. During evolution most of the cyanobacterial genes were transferred from the chloroplast to the nuclear genome. The rbcX gene, encoding an assembly chaperone required for Rubisco biosynthesis in cyanobacteria, was duplicated. Here we demonstrate that homologous eukaryotic chaperones (AtRbcX1 and AtRbcX2) demonstrate different affinities for the C-terminus of Rubisco large subunit and determine their crystal structures. METHODS Three-dimensional structures of AtRbcX1 and AtRbcX2 were resolved by the molecular replacement method. Equilibrium binding constants of the C-terminal RbcL peptide by AtRbcX proteins were determined by spectrofluorimetric titration. The binding mode of RbcX-RbcL was predicted using molecular dynamic simulation. RESULTS We provide crystal structures of both chaperones from Arabidopsis thaliana providing the first structural insight into Rubisco assembly chaperones form higher plants. Despite the low sequence homology of eukaryotic and cyanobacterial Rubisco chaperones the eukaryotic counterparts exhibit surprisingly high similarity of the overall fold to previously determined prokaryotic structures. Modeling studies demonstrate that the overall mode of the binding of RbcL peptide is conserved among these proteins. As such, the evolution of RbcX chaperones is another example of maintaining conserved structural features despite significant drift in the primary amino acid sequence. GENERAL SIGNIFICANCE The presented results are the approach to elucidate the role of RbcX proteins in Rubisco assembly in higher plants.

Structural Basis of GD2 Ganglioside and Mimetic Peptide Recognition by 14G2a Antibody.

Monoclonal antibodies targeting GD2 ganglioside (GD2) have recently been approved for the treatment of high risk neuroblastoma and are extensively evaluated in clinics in other indications. This study illustrates how a therapeutic antibody distinguishes between different types of gangliosides present on normal and cancer cells and informs how synthetic peptides can imitate ganglioside in its binding to the antibody. Using high resolution crystal structures we demonstrate that the ganglioside recognition by a model antibody (14G2a) is based primarily on an extended network of direct and water molecule mediated hydrogen bonds. Comparison of the GD2-Fab structure with that of a ligand free antibody reveals an induced fit mechanism of ligand binding. These conclusions are validated by directed mutagenesis and allowed structure guided generation of antibody variant with improved affinity toward GD2. Contrary to the carbohydrate, both evaluated mimetic peptides utilize a “key and lock” interaction mechanism complementing the surface of the antibody binding groove exactly as found in the empty structure. The interaction of both peptides with the Fab relies considerably on hydrophobic contacts however, the detailed connections differ significantly between the peptides. As such, the evaluated peptide carbohydrate mimicry is defined primarily in a functional and not in structural manner.

Small-molecule inhibitors of PD-1/PD-L1 immune checkpoint alleviate the PD-L1-induced exhaustion of T-cells.

Antibodies targeting the PD-1/PD-L1 immune checkpoint achieved spectacular success in anticancer therapy in the recent years. In contrast, no small molecules with cellular activity have been reported so far. Here we provide evidence that small molecules are capable of alleviating the PD-1/PD-L1 immune checkpoint-mediated exhaustion of Jurkat T-lymphocytes. The two optimized small-molecule inhibitors of the PD-1/PD-L1 interaction, BMS-1001 and BMS-1166, developed by Bristol-Myers Squibb, bind to human PD-L1 and block its interaction with PD-1, when tested on isolated proteins. The compounds present low toxicity towards tested cell lines and block the interaction of soluble PD-L1 with the cell surface-expressed PD-1. As a result, BMS-1001 and BMS-1166 alleviate the inhibitory effect of the soluble PD-L1 on the T-cell receptor-mediated activation of T-lymphocytes. Moreover, the compounds were effective in attenuating the inhibitory effect of the cell surface-associated PD-L1. We also determined the X-ray structures of the complexes of BMS-1001 and BMS-1166 with PD-L1, which revealed features that may be responsible for increased potency of the compounds compared to their predecessors. Further development may lead to the design of an anticancer therapy based on the orally delivered immune checkpoint inhibition.

Bioactive Macrocyclic Inhibitors of the PD-1/PD-L1 Immune Checkpoint

Blockade of the immunoinhibitory PD-1/PD-L1 pathway using monoclonal antibodies has shown impressive results with durable clinical antitumor responses. Anti-PD-1 and anti-PD-L1 antibodies have now been approved for the treatment of a number of tumor types, whereas the development of small molecules targeting immune checkpoints lags far behind. We characterized two classes of macrocyclic-peptide inhibitors directed at the PD-1/PD-L1 pathway. We show that these macrocyclic compounds act by directly binding to PD-L1 and that they are capable of antagonizing PD-L1 signaling and, similarly to antibodies, can restore the function of T-cells. We also provide the crystal structures of two of these small-molecule inhibitors bound to PD-L1. The structures provide a rationale for the checkpoint inhibition by these small molecules, and a description of their small molecule/PD-L1 interfaces provides a blueprint for the design of small-molecule inhibitors of the PD-1/PD-L1 pathway.

Investigation of Serine-Proteinase-Catalyzed Peptide Splicing in Analogues of Sunflower Trypsin Inhibitor 1 (SFTI-1)

Serine‐proteinase‐catalyzed peptide splicing was demonstrated in analogues of the trypsin inhibitor SFTI‐1: both single peptides and two‐peptide chains (C‐ and N‐terminal peptide chains linked by a disulfide bridge). In the second series, peptide splicing with catalytic amount of proteinase was observed only when formation of acyl–enzyme intermediate was preceded by hydrolysis of the substrate Lys–Ser peptide bond. Here we demonstrate that with an equimolar amount of the proteinase, splicing occurs in all the two‐peptide‐chain analogues. This conclusion was supported by high resolution crystal structures of selected analogues in complex with trypsin. We showed that the process followed a direct transpeptidation mechanism. Thus, the acyl–enzyme intermediate was formed and was immediately used for a new peptide bond formation; products associated with the hydrolysis of the acyl–enzyme were not observed. The peptide splicing was sequence‐ not structure‐specific.

Crystal structure of a low molecular weight activator Blm-pep with yeast 20S proteasome – insights into the enzyme activation mechanism

Proteasomes are responsible for protein turnover in eukaryotic cells, degrading short-lived species but also removing improperly folded or oxidatively damaged ones. Dysfunction of a proteasome results in gradual accumulation of misfolded/damaged proteins, leading to their aggregation. It has been postulated that proteasome activators may facilitate removal of such aggregation-prone proteins and thus prevent development of neurodegenerative disorders. However, the discovery of pharmacologically relevant compounds is hindered by insufficient structural understanding of the activation process. In this study we provide a model peptidic activator of human proteasome and analyze the structure-activity relationship within this novel scaffold. The binding mode of the activator at the relevant pocket within the proteasome has been determined by X-ray crystallography. This crystal structure provides an important basis for rational design of pharmacological compounds. Moreover, by providing a novel insight into the proteasome gating mechanism, our results allow the commonly accepted model of proteasome regulation to be revisited.

Crystal structure of ADP-dependent glucokinase from Methanocaldococcus jannaschii in complex with 5-iodotubercidin reveals phosphoryl transfer mechanism: Crystal Structure of ADPGK from M. jannaschii

ADP‐dependent glucokinase (ADPGK) is an alternative novel glucose phosphorylating enzyme in a modified glycolysis pathway of hyperthermophilic Archaea. In contrast to classical ATP‐dependent hexokinases, ADPGK utilizes ADP as a phosphoryl group donor. Here, we present a crystal structure of archaeal ADPGK from Methanocaldococcus jannaschii in complex with an inhibitor, 5‐iodotubercidin, d‐glucose, inorganic phosphate, and a magnesium ion. Detailed analysis of the architecture of the active site allowed for confirmation of the previously proposed phosphorylation mechanism and the crucial role of the invariant arginine residue (Arg197). The crystal structure shows how the phosphate ion, while mimicking a β‐phosphate group, is positioned in the proximity of the glucose moiety by arginine and the magnesium ion, thus providing novel insights into the mechanism of catalysis. In addition, we demonstrate that 5‐iodotubercidin inhibits human ADPGK‐dependent T cell activation‐induced reactive oxygen species (ROS) release and downstream gene expression, and as such it may serve as a model compound for further screening for hADPGK‐specific inhibitors.