Harald Kolmar

Decorating microbes: surface display of proteins on Escherichia coli

Bacterial surface display entails the presentation of recombinant proteins or peptides on the surface of bacterial cells. Escherichia coli is the most frequently used bacterial host for surface display and, as such, a variety of E. coli display systems have been described that primarily promote the surface exposure of peptides and small proteins. By contrast, display systems based on autotransporter proteins (ATs) and ice nucleation protein (INP) are excellent systems for the display of large and complex proteins, and are therefore of considerable biotechnological relevance. Here, we review recent advances in AT and INP-mediated display and their biotechnological applications. Additionally, we discuss several promising alternative display methods, as well as novel bacterial host organisms.

Biological diversity and therapeutic potential of natural and engineered cystine knot miniproteins.

Owing to their outstanding inherent thermal stability and proteolytic resistance, as well as their small size of only around 30 amino acid residues, cystine knot miniproteins are an attractive class of agents for the development of peptide-based pharmaceuticals. Many natural miniproteins already possess interesting pharmacological properties that can be used as starting point for further improvement by protein engineering. Cystine knot miniproteins, also known as knottins, are readily accessible by recombinant bacterial production or by solid phase chemical synthesis. Potent and selective knottins with predefined binding characteristics were obtained by rational protein design as well as by combinatorial library screening. Owing to their proteolytic stability and relatively high rates of intestinal uptake, the oral administration of miniproteins seems to be within reach. With the first engineered knottin successfully applied for tumor imaging and several others being already marketed as analgesic or in preclinical and clinical development, it can be expected that more tailor-made diagnostic and therapeutic cystine knot miniproteins will follow over the next years.

The crystal structure of SdsA1, an alkylsulfatase from Pseudomonas aeruginosa, defines a third class of sulfatases

Pseudomonas aeruginosa is both a ubiquitous environmental bacterium and an opportunistic human pathogen. A remarkable metabolic versatility allows it to occupy a multitude of ecological niches, including wastewater treatment plants and such hostile environments as the human respiratory tract. P. aeruginosa is able to degrade and metabolize biocidic SDS, the detergent of most commercial personal hygiene products. We identify SdsA1 of P. aeruginosa as a secreted SDS hydrolase that allows the bacterium to use primary sulfates such as SDS as a sole carbon or sulfur source. Homologues of SdsA1 are found in many pathogenic and some nonpathogenic bacteria. The crystal structure of SdsA1 reveals three distinct domains. The N-terminal catalytic domain with a binuclear Zn2+ cluster is a distinct member of the metallo-β-lactamase fold family, the central dimerization domain ensures resistance to high concentrations of SDS, whereas the C-terminal domain provides a hydrophobic groove, presumably to recruit long aliphatic substrates. Crystal structures of apo-SdsA1 and complexes with substrate analog and products indicate an enzymatic mechanism involving a water molecule indirectly activated by the Zn2+ cluster. The enzyme SdsA1 thus represents a previously undescribed class of sulfatases that allows P. aeruginosa to survive and thrive under otherwise bacteriocidal conditions.

Alternative binding proteins: Biological activity and therapeutic potential of cystine‐knot miniproteins

Cystine‐knot miniproteins are members of a large family of small proteins that are defined by a common structural scaffold which is stabilized by three intramolecular disulfide bonds. Cystine‐knot miniproteins display a broad spectrum of therapeutically useful natural biological activities and several family members are marketed as therapeutics or are in clinical development. Because of their extraordinary intrinsic chemical and proteolytic stability they provide promising scaffolds for the introduction of therapeutically relevant functionalities. Several successful engineering efforts have been reported to generate miniproteins with novel activities by rational design via functional loop grafting or by directed evolution via screening of scaffold‐constrained random libraries. Owing to their small size they are amenable to recombinant as well as to chemical routes of synthesis, which opens up new avenues in optimizing biological activity, specificity and bioavailability by site‐specific modification, introduction of non‐natural amino acids or chemical conjugation.

The potential of cystine-knot microproteins as novel pharmacophoric scaffolds in oral peptide drug delivery

Within this study, the potential of three clinically relevant microproteins (SE–AG–AZ, SE–EM and SE–EP) with cystine-knot architecture as pharmacophoric scaffolds for oral peptide delivery was investigated. Cystine-knot microproteins (CKM) were analysed regarding their stability towards the most important gastrointestinal secreted and membrane bound proteases in physiological concentrations. In addition, their permeation behaviour through freshly excised rat intestinal mucosa as well as important parameters such as aggregation behaviour, stability in rat plasma and isoelectric point were evaluated and compared to the properties of the model peptide drugs bacitracin and insulin. Aggregation studies indicate that under physiological conditions between 25 and 70% of the CKMs occur as monomers, whereas the rest forms di- and trimers. Pepsin and elastase cause no or only minor degradation to CKMs, whereas trypsin and chymotrypsin degrade CKMs extensively. Removing the theoretical chymotrypsin cleavage site from a CKM, however, led to stabilization towards this protease. Two of the three evaluated CKMs are stable against membrane bound proteases. Papp values were determined to be 5.96 ± 0.98 × 10− 6 and 6.63 ± 0.47 × 10− 6 cm/s. In conclusion, this study indicates that CKM are promising novel pharmacophoric scaffolds for oral peptide delivery.

A generic system for the Escherichia coli cell-surface display of lipolytic enzymes.

EstA is an outer membrane‐anchored esterase from Pseudomonas aeruginosa. An inactive EstA variant was used as an anchoring motif for the Escherichia coli cell‐surface display of lipolytic enzymes. Flow cytometry analysis and measurement of lipase activity revealed that Bacillus subtilis lipase LipA, Fusarium solani pisi cutinase and one of the largest lipases presently known, namely Serratia marcescens lipase were all efficiently exported by the EstA autotransporter and also retained their lipolytic activities upon cell surface exposition. EstA provides a useful tool for surface display of lipases including variant libraries generated by directed evolution thereby enabling the identification of novel enzymes with interesting biological and biotechnological ramifications.

Triazole bridge: disulfide-bond replacement by ruthenium-catalyzed formation of 1,5-disubstituted 1,2,3-triazoles.

About one fourth of the peptidic macromolecular structures deposited in the protein data base (PDB) contain at least one disulfide bridge. In nature, disulfide bonds are formed in a milieu where oxidizing conditions prevail, for example, on the cell surface or in the extracellular matrix. Many proteins benefit from disulfide contributions to their conformational stability. In particular, the defined tertiary folding of oligopeptides smaller than 30 residues essentially relies on macrocyclization through the cystine motif because of the restricted number of noncovalent intramolecular interactions available. Moreover, formation of the disulfide pattern results in structural rigidity of the peptidic framework, as for example, in the family of cystine knot miniproteins, leading to conformationally constrained scaffolds with extraordinary thermal stability and resistance against proteolytic degradation. Hence, the discovery and development of disulfidebridged peptides suitable for diagnostic and therapeutic applications remains a field of intense research. The in-vitro generation of disulfide bonds in peptides is usually achieved post-synthetically and mediated by DMSO, air oxygen, or other oxidizing agents. Although this reaction step can be achieved under relatively mild conditions in solution, it remains one of the most demanding obstacles towards high-yield peptide synthesis, especially for disulfiderich species in which the controlled regiospecific formation of several disulfide bonds is not trivial to control. In addition, to suppress unwanted intermolecular reactions of the thiol groups of individual peptides, oxidative folding usually has to be conducted in highly diluted solutions. In spite of the use of gluthathione-based redox buffers, polymer-supported oxidation systems, macrocyclization on the solid support and/or orthogonal protecting groups, control over the topology of the disulfide bridges formed is still a challenge. 5] In view of these difficulties and to improve the redox stability of bridged peptides, several routes towards synthetic disulfide surrogates have been developed. Straightforward approaches usually employ thioether, olefin, or alkane-based isosters. However, cystathione bridges require multiple synthetic steps and careful choice of orthogonal protection, and dicarba bridges give cis/trans isomers during ring-closing metathesis (RCM). Only an additional purification step or the subsequent palladium-catalyzed hydrogenation of the unsaturated species to the corresponding alkane leads to a construct with defined configuration. In 2004, Meldal et al. described the utility of copper(I)catalyzed azide–alkyne cycloaddition (CuAAC) for a triazole-based disulfide replacement. Owing to the compelling characteristics of this prototypic “click” reaction, it has been extensively applied in peptide chemistry exploiting the almost perfect orthogonality to side-chain reactivities. The introduction of 1,4-disubstituted 1,2,3-triazoles into peptides has also been used to mimic and rigidify conformations of the amide backbone. Moreover, a variety of examples of CuAAC-based macrocyclizations of peptides in solution and on solid supports has been reported. Using the same azideand alkyne-functionalized buidling blocks, 1,5-disubstituted 1,2,3-triazoles can be generated in the ruthenium(II)-catalyzed variant (RuAAC) of the CuAAC. This reaction expands the range of peptidomimetic structures selectively accessible from the same precursor and having different biological activities governed by the architecture of the incorporated triazole. To our knowledge, 1,5-disubstitiuted 1,2,3-triazoles have not been taken into consideration as disulfide mimics to date. Herein, we report the facile introduction of 1,4and 1,5disubstituted 1,2,3-triazoles into a monocyclic variant of the sunflower trypsin inhibitor-I (SFTI-1[1,14], 1; Figure 1) and show that the macrocyclic peptidomimeticum 2 with the “1,5” substitution pattern retains nearly full biological activity in contrast to the “1,4” variants 3 and 4. The choice of 1 as the model peptide for the investigation of triazole-based disulfide replacements had several reasons. SFTI-1 is a small, though very potent, inhibitor of trypsin. Therefore, the influence of different modes of macrocyclization on the bioactivity of the corresponding synthetic variant can be routinely examined by serine protease inhibition assays. [*] M. Empting, Dr. O. Avrutina, Dr. R. Meusinger, S. Fabritz, M. Reinwarth, Prof. Dr. H. Kolmar Clemens-Sch pf-Institut f r Organische Chemie und Biochemie Technische Universit t Darmstadt Petersenstrasse 22, 64287 Darmstadt (Germany) Fax: (+49)6151-16-5399 E-mail: kolmar@biochemie-tud.de Homepage: http://www.chemie.tu-darmstadt.de/kolmar

Inhibition of platelet aggregation by grafting RGD and KGD sequences on the structural scaffold of small disulfide-rich proteins

Disintegrins represent a group of disulfide-rich peptides ranging in size from 41 to over 80 residues and are antagonists of several integrin receptors. Disintegrins containing an RGD or KGD sequence are potent inhibitors of platelet aggregation as they block the binding of fibrinogen to αIIbβ3 integrin. The high affinity binding to αIIbβ3 in comparison to short linear peptides has been attributed to the localisation of the RGD or KGD sequence within a defined three-dimensional structure. Cystine knot microproteins are members of another family of small disulfide-rich peptides that consist of only 28–40 amino acid residues. They display numerous biological activities depending on the peptide sequence of loop regions that are fixed on a structural scaffold that is stabilised by three knot-forming disulfide bonds. In the present study we grafted RGD and KGD containing peptide sequences with seven and 11 amino acids, respectively, into two cystine knot microproteins, the trypsin inhibitor EETI-II and the melanocortin receptor binding domain of the human agouti-related protein AGRP, as well as into the small disintegrin obtustatin. The engineered proteins were much more potent to inhibit the fibrinogen binding, αIIbβ3 activation and platelet aggregation when compared to the grafted peptides. Differences that were observed between the engineered proteins indicate the importance of the structural scaffold and the amino acids neighbouring the grafted peptide sequences.

Sequence requirements of the GPNG beta-turn of the Ecballium elaterium trypsin inhibitor II explored by combinatorial library screening.

The Ecballium elaterium trypsin inhibitor II (EETI-II) contains 28 amino acids and three disulfides forming a cystine knot. Reduced EETI-II refolds spontaneously and quantitatively in vitro and regains its native structure. Due to its high propensity to form a reverse turn, the GPNG sequence of segment 22–25 comprising a β-turn in native EETI-II is a possible candidate for a folding initiation site. We generated a molecular repertoire of EETI-II variants with variegated 22–25 tetrapeptide sequences and presented these proteins on the outer membrane ofEscherichia coli cells via fusion to the Igaβautotransporter. Functional trypsin-binding variants were selected by combination of magnetic and fluorescence-activated cell sorting. At least 1–5% of all possible tetrapeptide sequences were compatible with formation of the correct three disulfides. Occurrence of amino acid residues in functional variants is positively correlated with their propensity to be generally found in β-turns. The folding pathway of two selected variants, EETI-βNEDE and EETI-βTNNK, was found to be indistinguishable from EETI-II and occurs through formation of a stable 2-disulfide intermediate. Substantial amounts of misfolded byproducts, however, were obtained upon refolding of these variants corroborating the importance of the wild type EETI-II GPNG sequence to direct quantitative formation of the cystine knot architecture.

Membrane insertion of the bacterial signal transduction protein ToxR and requirements of transcription activation studied by modular replacement of different protein substructures.

The Vibrio cholerae protein ToxR is an integral membrane protein that acts as a transcription activator in response to environmental signals; it controls expression of toxin genes ctxA and ctxB, along with a variety of other genes related to pathogenicity. Here it is shown that: (i) ToxR has a modular architecture and that activation of transcription starting at the ctx promoter depends strictly on dimerization of the periplasmic ToxR domain; (ii) the transmembrane (TM) region of ToxR is sufficient as a topogenic signal but not for stable membrane anchoring of the protein; (iii) the TM region has no special function in signal transduction and (iv) a proline residue located within the TM region minimizes background transcription activation, most plausibly by reducing TM‐TM interaction. Possible applications of ToxR as a technical tool for analysing protein‐protein interactions between pairs of arbitrary TM domains are discussed.