Jacek Otlewski

Canonical protein inhibitors of serine proteases.

Serine proteases and their natural protein inhibitors are among the most intensively studied protein complexes. About 20 structurally diverse inhibitor families have been identified, comprising α-helical, β sheet, and α/β proteins, and different folds of small disulfide-rich proteins. Three different types of inhibitors can be distinguished based on their mechanism of action: canonical (standard mechanism) and non-canonical inhibitors, and serpins. The canonical inhibitors bind to the enzyme through an exposed convex binding loop, which is complementary to the active site of the enzyme. The mechanism of inhibition in this group is always very similar and resembles that of an ideal substrate. The non-canonical inhibitors interact through their N-terminal segment. There are also extensive secondary interactions outside the active site, contributing significantly to the strength, speed, and specificity of recognition. Serpins, similarly to the canonical inhibitors, interact with their target proteases in a substrate-like manner; however, cleavage of a single peptide bond in the binding loop leads to dramatic structural changes.

The refined 2.0 Å X-ray crystal structure of the complex formed between bovine β-trypsin and CMTI-I, a trypsin inhibitor from squash seeds (Cucurbita maxima) Topological similarity of the squash seed inhibitors with the carboxypeptidase A inhibitor from potatoes

The stoichiometric complex formed between bovine β‐trypsin and the Cucurbita maxima trypsin inhibitor I (CMTI‐I) was crystallized and its X‐ray crystal structure determined using Patterson search techniques. Its structure has been crystallographically refined to a final R value of 0.152 (6.0 — 2.0 Å). CMTI‐I is of ellipsoidal shape; it lacks helices or β‐sheets, but consists of turns and connecting short polypeptide stretches. The disulfide pairing is CYS‐3I‐20I, Cys‐10I‐22I and Cys‐16I‐28I. According to the polypeptide fold and disulfide connectivity its structure resembles that of the carboxypeptidase A inhibitor from potatoes. Thirteen of the 29 inhibitor residues are in direct contact with trypsin; most of them are in the primary binding segment Val‐2I (P4) — Glu‐9I (P4′) which contains the reactive site bond Arg‐5I — Ile‐6I and is in a conformation observed also for other serine proteinase inhibitors.

Determination of the complete three-dimensional structure of the trypsin inhibitor from squash seeds in aqueous solution by nuclear magnetic resonance and a combination of distance geometry and dynamical simulated annealing.

The complete three-dimensional structure of the trypsin inhibitor from seeds of the squash Cucurbita maxima in aqueous solution was determined on the basis of 324 interproton distance constraints, 80 non-nuclear Overhauser effect distances, and 22 hydrogen-bonding constraints, supplemented by 27 phi backbone angle constraints derived from nuclear magnetic resonance measurements. The nuclear magnetic resonance input data were converted to the distance constraints in a semiquantitative manner after a sequence specific assignment of 1H spectra was obtained using two-dimensional nuclear magnetic resonance techniques. Stereospecific assignments were obtained for 17 of the 48 prochiral centers of the squash trypsin inhibitor using the floating chirality assignment introduced at the dynamical simulated annealing stage of the calculations. A total of 34 structures calculated by a hybrid distance geometry-dynamical simulated annealing method exhibit well-defined positions for both backbone and side-chain atoms. The average atomic root-mean-square difference between the individual structures and the minimized mean structure is 0.35(+/- 0.08) A for the backbone atoms and 0.89(+/- 0.17) A for all heavy atoms. The precision of the structure determination is discussed and correlated to the experimental input data.

Aptamers: Molecules of great potential

Aptamers emerged over 20 years ago as a class of nucleic acids able to recognize specific targets. Today, aptamer-related studies constitute a large and important field of biotechnology. Functional oligonucleotides have proved to be a versatile tool in biomedical research due to the ease of synthesis, a wide range of potentially recognized molecular targets and the simplicity of selection. Similarly to antibodies, aptamers can be used to detect or isolate specific molecules, as well as to act as targeting and therapeutic agents. In this review we present different approaches to aptamer application in nanobiotechnology, diagnostics and medicine.

Crystal Structure of Vigna radiata Cytokinin-Specific Binding Protein in Complex with Zeatin

The cytosolic fraction of Vigna radiata contains a 17-kD protein that binds plant hormones from the cytokinin group, such as zeatin. Using recombinant protein and isothermal titration calorimetry as well as fluorescence measurements coupled with ligand displacement, we have reexamined the Kd values and show them to range from ∼10−6 M (for 4PU30) to 10−4 M (for zeatin) for 1:1 stoichiometry complexes. In addition, we have crystallized this cytokinin-specific binding protein (Vr CSBP) in complex with zeatin and refined the structure to 1.2 Å resolution. Structurally, Vr CSBP is similar to plant pathogenesis-related class 10 (PR-10) proteins, despite low sequence identity (<20%). This unusual fold conservation reinforces the notion that classic PR-10 proteins have evolved to bind small-molecule ligands. The fold consists of an antiparallel β-sheet wrapped around a C-terminal α-helix, with two short α-helices closing a cavity formed within the protein core. In each of the four independent CSBP molecules, there is a zeatin ligand located deep in the cavity with conserved conformation and protein–ligand interactions. In three cases, an additional zeatin molecule is found in variable orientation but with excellent definition in electron density, which plugs the entrance to the binding pocket, sealing the inner molecule from contact with bulk solvent.

PDZ Tandem of Human Syntenin: Crystal Structure and Functional Properties

Syntenin, a 33 kDa protein, interacts with several cell membrane receptors and with merlin, the product of the causal gene for neurofibromatosis type II. We report a crystal structure of the functional fragment of human syntenin containing two canonical PDZ domains, as well as binding studies for full-length syntenin, the PDZ tandem, and isolated PDZ domains. We show that the functional properties of syntenin are a result of independent interactions with target peptides, and that each domain is able to bind peptides belonging to two different classes: PDZ1 binds peptides from classes I and III, while PDZ2 interacts with classes I and II. The independent binding of merlin by PDZ1 and syndecan-4 by PDZ2 provides direct evidence for the coupling of syndecan-mediated signaling to actin regulation by merlin.

Trypsin Specificity as Elucidated by Lie Calculations, X-Ray Structures, and Association Constant Measurements

The variation in inhibitor specificity for five different amine inhibitors bound to CST, BT, and the cold‐adapted AST has been studied by use of association constant measurements, structural analysis of high‐resolution crystal structures, and the LIE method. Experimental data show that AST binds the 1BZA and 2BEA inhibitors 0.8 and 0.5 kcal/mole more strongly than BT. However, structural interactions and orientations of the inhibitors within the S1 site have been found to be virtually identical in the three enzymes studied. For example, the four water molecules in the inhibitor‐free structures of AST and BT are channeled into similar positions in the S1 site, and the nitrogen atom(s) of the inhibitors are found in two cationic binding sites denoted Position1 and Position2. The hydrophobic binding contributions for all five inhibitors, estimated by the LIE calculations, are also in the same order (−2.1 ± 0.2 kcal/mole) for all three enzymes. Our hypothesis is therefore that the observed variation in inhibitor binding arises from different electrostatic interactions originating from residues outside the S1 site. This is well illustrated by AST, in which Asp 150 and Glu 221B, despite some distance from the S1 binding site, lower the electrostatic potential of the S1 site and thus enhance substrate binding. Because the trends in the experimentally determined binding energies were reproduced by the LIE calculations after adding the contribution from long‐range interactions, we find this method very suitable for rational studies of protein–substrate interactions.

Current methods for the synthesis of homogeneous antibody-drug conjugates.

Development of efficient and safe cancer therapy is one of the major challenges of the modern medicine. Over the last few years antibody-drug conjugates (ADCs) have become a powerful tool in cancer treatment with two of them, Adcetris® (brentuximab vedotin) and Kadcyla® (ado-trastuzumab emtansine), having recently been approved by the Food and Drug Administration (FDA). Essentially, an ADC is a bioconjugate that comprises a monoclonal antibody that specifically binds tumor surface antigen and a highly potent drug, which is attached to the antibody via either cleavable or stable linker. This approach ensures specificity and efficacy in fighting cancer cells, while healthy tissues remain largely unaffected. Conventional ADCs, that employ cysteine or lysine residues as conjugation sites, are highly heterogeneous. This means that the species contain various populations of the ADCs with different drug-to-antibody ratios (DARs) and different drug load distributions. DAR and drug-load distribution are essential parameters of ADCs as they determine their stability and efficacy. Therefore, various drug-loaded forms of ADCs (usually from zero to eight conjugated molecules per antibody) may have distinct pharmacokinetics (PK) in vivo and may differ in clinical performance. Recently, a significant progress has been made in the field of site-specific conjugation which resulted in a number of strategies for synthesis of the homogeneous ADCs. This review describes newly-developed methods that ensure homogeneity of the ADCs including use of engineered reactive cysteine residues (THIOMAB), unnatural amino acids, aldehyde tags, enzymatic transglutaminase- and glycotransferase-based approaches and novel chemical methods. Furthermore, we briefly discuss the limitation of these methods emphasizing the need for further improvement in the ADC design and development.

Variability of the canonical loop conformations in serine proteinases inhibitors and other proteins

Canonical loops of protein inhibitors of serine proteinases occur in proteins having completely different folds. In this article, conformations of the loops have been analyzed for inhibitors belonging to 10 structurally different families. Using deviation in Cα‐Cα distances as a criterion for loop similarity, we found that the P3‐P3′ segment defines most properly the length of the loop. When conformational differences among loops of individual inhibitors were compared using root mean square deviation (rmsd) in atomic coordinates for all main chain atoms (Δr method) and rmsd operating in main chain torsion angles (Δt method), differences of up to 2.1 Å and 72.3°, respectively, were observed. Such large values indicate significant conformational differences among individual loops. Nevertheless, the overall geometry of the inhibitor‐proteinase interaction is very well preserved, as judged from the similarity of Cα‐Cα distances between Cα of catalytic Ser and Cα of P3‐P3′ residues in various enzyme‐inhibitor complexes. The mode of interaction is very well preserved both in the chymotrypsin and subtilisin families, as distances calculated for subtilisin‐inhibitor complexes are almost always within the range of those for chymotrypsin‐inhibitor complexes. Complex formation leads to conformational changes of up to 160° for χ1 angle. Side chains of residue P1 and P2′ adopt in different complexes a similar orientation (χ1 angle = −60° and −180°, respectively). To check whether the canonical conformation can be found among non–proteinase‐inhibitor Brookhaven Protein Data Bank structures, two selection criteria—the allowed main chain dihedral angles and Cα‐Cα distances for the P3‐P3′ segment—were applied to all these structures. This procedure detected 132 unique hexapeptide segments in 121 structurally and functionally unrelated proteins. Partial preferences for certain amino acids occurring at particular positions in these hexapeptides could be noted. Further restriction of this set to hexapeptides with a highly exposed P1 residue side chain resulted in 40 segments. The possibility of complexes formation between these segments and serine proteinases was ruled out in molecular modeling due to steric clashes. Several structural features that determine the canonical conformation of the loop both in inhibitors and in other proteins can be distinguished. They include main chain hydrogen bonds both within the P3‐P3′ segment and with the scaffold region, P3‐P4 and P3′‐P4′ hydrophobic interactions, and finally either hydrophobic or polar interactions involving the P1′ residue. Proteins 32:459–474, 1998.

Nuclear magnetic resonance solution and X-ray structures of squash trypsin inhibitor exhibit the same conformation of the proteinase binding loop.

A comparison of the solution nuclear magnetic resonance (n.m.r.) structures of squash trypsin inhibitor from seeds of the squash Cucurbita maxima with the X-ray structure of a trypsin complex of the inhibitor shows that the n.m.r. and X-ray structures are similar in terms of the global folding and secondary structure. The average atomic root-mean-square difference between the 36 n.m.r. structures on the one hand and the X-ray structure is 0.96 A for the backbone atoms and 1.95 A for all heavy atoms. The n.m.r. and X-ray structures exhibit extremely similar conformations of the primary proteinase binding loop. Despite the overall similarity, there are small differences between the mean computed structure and the X-ray structure. The n.m.r. structures have slightly different positions of the segments from residues 16 to 18, and 24 and 25. The n.m.r. results show that the inclusion of stereospecific assignments and precise distance constraints results in a significant improvement in the definition of the n.m.r. structure, making possible a detailed analysis of the local conformations in the protein.