Cosmix-plexing: a novel recombinatorial approach for evolutionary selection from combinatorial libraries.

The efficiency of existing combinatorial biological library methods has been moderate in terms of the success rates, the affinities of the ligands selected and the time and effort involved in trying to optimize the initial leads. Although mimicking natural evolution, existing strategies take little notice of the importance of recombination within a selected population to generate increased diversity. We present an overview of our recent progress which has resulted in the successful development of such a strategy, which we designate cosmix-plexing. We incorporate recombination as a central feature in obtaining high success rates and high affinities, even for short monomer peptides, in a very short time. The method uses type II restriction enzymes to re-assort small hypervariable DNA cassettes from an intermediate pre-selected population (e.g. from a phagemid display library), while maintaining the original open-reading frame. Since, in the naive library, each cassette contains all possible combinations of the polypeptide sequences it encodes, much longer regions can be optimized than was possible with methods which depend on a simple selection from the naive library. Short peptides can now be rapidly selected, which exhibit the same, or higher, specificity and affinity for a defined target molecule, than (say) an antibody or even the natural ligand.

Detection of regions in the MC1 receptor of importance for the selectivity of the MC1 receptor super-selective MS04/MS05 peptides.

We have investigated the ability of our earlier identified MS04-MS05 MSH-peptide analogues to bind to chimeric MC1-MC3 receptors. While the MS04 and MS05 peptides bind with nanomolar and sub-nanomolar affinities to the wild type MC1 receptor, they bind only with micromolar affinities for the wild type MC3 receptor, thus being the hitherto most MC1 receptor selective ligands. Upon exchanging portions involving transmembrane regions TM1, TM2-3, and TM6-7 of the MC1 receptor with corresponding portions of the MC3 receptor both of these peptides showed major losses of affinities. By contrast exchanges involving TM4-5 did not appreciably affect the affinity of either MS04 or MS05. Our data suggest that the binding pocket for the MS04-MS05 MSH-peptides is located between TM1-3 and TM6-7 of the melanocortin receptors.

New highly specific agonistic peptides for human melanocortin MC(1) receptor.

A peptide with very high specificity for the human melanocortin MC(1) receptor identified by phage display was used as a lead for the design of new peptides. Two new peptides, MS05 and MS09, were synthesized and found to bind with sub-nanomolar affinities to the MC(1) receptor. Both these peptides showed strong agonistic activity at the MC(1) receptor. The MS05 was the most MC(1) receptor selective as it showed virtually no binding affinity for the MC(4) and MC(5) receptors and only micromolar affinity for the MC(3) receptor. The selectivity and potency of the new peptides make them potent tools for studies of MC(1) receptors, as well as novel potential candidate drugs for the treatment of inflammatory conditions.

Chimeric melanocortin MC1 and MC3 receptors: identification of domains participating in binding of melanocyte-stimulating hormone peptides.

The melanocortin receptors MC1 and MC3 are G protein-coupled receptors that have substantial structural similarities and bind melanocyte peptides but with different affinity profiles. We constructed a series of chimeric MC1/MC3 receptors to identify the epitopes that determine their selectivities for natural melanocyte peptides and synthetic analogues. The chimeric constructs were made by a polymerase chain reaction that used identical regions in or just outside transmembranes (TM) 1, 4, and 6 and divided the receptors into four segments. Saturation and competition studies on the expressed chimeric proteins indicate that TM1, TM2, TM3, and TM7 are involved in the subtype-specific binding of melanocyte peptides to these receptors. The results support the hypothesis that TM4 and TM5 may not contribute to the ligand-binding specificity of the MC receptors. This is the first report to describe the subtype-specific hormone-binding domains of the melanocortin receptor family.

Phage display selection on whole cells yields a peptide specific for melanocortin receptor 1.

A phage display system for the selection of peptides binding to heterologously expressed human melanocortin receptor 1 on the surface of insect cells has been established. It could be shown that phage particles displaying the natural ligand alpha-melanocyte-stimulating hormone bind selectively to cells expressing this receptor and that these phages exhibit biological activity on mouse B16F1 melanoma cells. Insect cells were superior to other cell lines tested and have been used to select binders from a small library, in which critical determinants (Phe7-Arg8-Trp9) were kept, whereas the flanking regions where allowed to variate freely. One peptide displaying little similarity with native hormone was found that binds to the receptor also in its free form with an affinity of 7 nM. It showed a remarkable selectivity for this receptor, because it binds to the other melanocortin receptor subtypes with a maximum affinity of 21 microM. This is the first time phage display has been used successfully with G-protein-coupled receptors lacking an extracellular binding domain.

Deletions of the N-terminal regions of the human melanocortin receptors.

The non-homologous N-terminal regions of four human melanocortin (MC) receptors were truncated in order to investigate their putative participation in ligand binding. Eleven constructs were made, where different numbers of residues from the N terminus were deleted. These constructs were used for transient expression experiments in COS cells and analysed by ligand binding. The results show that 27, 25, 28, and 20 amino acids could be deleted from the N terminus of the human MC1, MC3, MC4 and MC5 receptors, respectively, including all potential N-terminal glycosylation sites in the MC1 and the MC4 receptors, without affecting ligand binding or expression levels. The results indicate that the N-terminal regions of the human MC1, MC3, MC4 and MC5 receptors, do not play an important role for the ligand binding properties of these receptors.

Characterisation of D117A and H260A mutations in the melanocortin 1 receptor.

Recent site directed mutagenesis studies on the melanocortin 1 (MC1) receptor have indicated the importance of D117 and H260 amino acid residues for the binding of alpha-MSH (melanocyte stimulating hormone). Here, we report the testing of 12 cyclic and linear MSH peptides on the D117A and H260A mutant receptors. Moreover, we constructed a double mutant which displayed a major loss in affinity for [Nle4, D-Phe7]alpha-MSH. Our new data of His6 and Phe7 substituted MSH peptides are compared with previous results and the hypothesis of putative interactions of D117 and H260 with single amino acids in the MSH peptide. Our conclusions are that the D117A and the H260A mutations may cause conformational changes in the receptor which can not be linked to any specific amino acid in the MSH-peptides.

Binding of cyclic and linear MSH core peptides to the melanocortin receptor subtypes.

We report here the binding of 5-, 6- and 7-amino-acid-long linear and cyclic core peptides of MSH (melanocyte-stimulating hormone) to cells transiently expressing the human melanocortin MC1, MC3, MC4 and MC5 receptors. The results show that, in contrast to the natural peptides, the core peptides did not differentiate between the melanocortin MC3 and MC4 receptors. All tested cyclic peptides had much lower affinities than their corresponding linear homologues. Interestingly, the relative loss of binding due to the cyclisation did not change as the ring size decreased. Therefore, decreasing the ring size does not seem to force the peptide into a more unfavourable conformation.

Evidence indicating that the TM4, EL2, and TM5 of the melanocortin 3 receptor Do not participate in ligand binding.

The TM4, EL2 and TM5 show low amino acid homology within the MC receptor family. Three mutants of the human MC3 receptor were created in order to investigate the participation of these regions in ligand binding. The TM4, EL2 and TM5 were separately changed by multiple mutagenesis so that their amino acid sequences became identical with the human MC1 receptor. The mutants were expressed in COS cells and they bound peptide ligands in the same fashion as the wild type MC3 receptor clone. Our results indicate that the amino acids that were mutated in the MC3 receptor do not affect the binding of MSH peptides. The data provide further evidence, that the mutated regions may not participate at all in ligand binding, as indicated by modelling experiments and homology comparison.

Alternative translation initiation codon for the human melanocortin MC3 receptor does not affect the ligand binding.

The genomic DNA for the human melanocortin MC3 receptor indicates an unusually long N-terminus. Two possible translation initiation sites, the one originally proposed and one alternate 111 bp downstream, were mutated. For a third mutant the DNA between these initiation sites was deleted. All mutants were expressed in COS (CV-1 Origin, SV40) cells in the same level, and they bound peptide hormones in the same fashion, as did the wild type clone. The data obtained indicate that both sites can function as the sole translation initiation sites of the human clone and that the proposed N-terminus of the human melanocortin MC3 receptor is not important for the ligand binding of the receptor.

The active site of Trichoderma reesei cellobiohydrolase II: the role of tyrosine 169.

Trichoderma reesei cellobiohydrolase II (CBHII) is an exoglucanase cleaving primarily cellobiose units from the non-reducing end of cellulose chains. The beta-1,4 glycosidic bond is cleaved by acid catalysis with an aspartic acid, D221, as the likely proton donor, and another aspartate, D175, probably ensuring its protonation and stabilizing charged reaction intermediates. The catalytic base has not yet been identified experimentally. The refined crystal structure of CBHII also shows a tyrosine residue, Y169, located close enough to the scissile bond to be involved in catalysis. The role of this residue has been studied by introducing a mutation Y169F, and analysing the kinetic and binding behavior of the mutated CBHII. The crystal structure of the mutated enzyme was determined to 2.0 A resolution showing no changes when compared with the structure of native CBHII. However, the association constants of the mutant enzyme for cellobiose and cellotriose are increased threefold and for 4-methylumbelliferyl cellobioside over 50-fold. The catalytic constants towards cellotriose and cellotetraose are four times lower for the mutant. These data suggest that Y169, on interacting with a glucose ring entering the second subsite in a narrow tunnel, helps to distort the glucose ring into a more reactive conformation. In addition, a change in the pH activity profile was observed. This indicates that Y169 may have a second role in the catalysis, namely to affect the protonation state of the active site carboxylates, D175 and D221.

Expression of functional melanocortin 1 receptors in insect cells.

We expressed epitope-tagged human melanocortin 1 receptor (MC1R) in insect cells using two different recombinant baculovirus constructs; one of which encoded MC1R with an N-terminal Flag epitope and a C-terminal polyHis tag, while the other encoded the MC1R with a C-terminal Myc tag. The constructs were used to infect Sf9 insect cells. For both constructs, immunoblotting with tag-specific antibodies demonstrated the presence of the receptor in the infected cells. The infected Sf9 cells were characterized by radioligand binding using [125I][Nle4,D-Phe7]alpha-MSH. Both saturation and competition analysis, using alpha-, beta-, and gamma 1-MSH on the tagged MC1R expressed in the insect cells, gave binding constants and potency orders that were undistinguishable from those obtained on MC1R expressed in COS cells. The expression level obtained (in the order of pmoles of binding sites per mg of protein) will now facilitate attempts to purify the receptor. This is the first report that demonstrates a functional expression of recombinant melanocortin receptor in nonmammalian cells.

Highly effective protease inhibitors from variants of human pancreatic secretory trypsin inhibitor (hPSTI): an assessment of 3-D structure-based protein design.

The results of a protein design project are used to compare different predictive strategies with respect to protein-protein interactions. We have been able to generate variants of human pancreatic secretory trypsin inhibitor (hPSTI) optimized with respect to the affinity and specificity for human leukocyte elastase relative to trypsin and chymotrypsin, and in particular chymotrypsin. The extremely strong and specific human leukocyte elastase inhibitors were thus developed in three rounds of mutagenesis and two rounds of 3-D modelling; only 24 variants in total were synthesized, although variations at seven different amino acid positions were involved (i.e. from 20(7) possible variants). An excellent elastase inhibitor could be designed with the minimum of two amino acid exchanges. The value of structural modelling and actual structure determination is discussed in the light of the experimental results of the designed protein variants and the results of tertiary structure determinations of the free variant and the inhibitor-protease complex. Particular reference is given to the strategy to be followed in protein design projects in general and to the development of protease inhibitors in particular.

In vivo biological activity of retinoids partially correlates to their affinity to recombinant retinoic-acid receptor alpha and recombinant-cellular retinoic-acid-binding protein I.

Several known and some new retinoids were synthesized and their in vivo activity was investigated by an assay, based on induction of alkaline phosphatase in P19 teratocarcinoma cells, human prostate carcinoma cells and primary cultures of neonatal rat heart cells. The assay used in this study was found to be reproducible and useful for rapid screening of retinoids for biological activity. Two newly synthesized compounds exhibit high biological activity. The biological potency of the compounds was compared to their ability to bind to recombinant retinoic-acid receptor alpha and to cellular retinoic-acid-binding protein I determined by Charsorb-binding assay. mRNA of both retinoic-acid-binding proteins could be detected in the three cell lines investigated. As expected from the number of different retinoic-acid receptors, the results suggest that retinoids do not need to bind retinoic-acid receptor alpha nor cellular retinoic-acid-binding protein I in order to exhibit biological activity, but most retinoids investigated show a clear correlation between binding to these proteins and their biological activity.

Three-dimensional structure of a recombinant variant of human pancreatic secretory trypsin inhibitor (Kazal type).

A modified version of the human pancreatic trypsin inhibitor (PSTI), generated in a protein-design project, has been crystallized in spacegroup P4(3) with lattice constants a = 40.15 A, c = 33.91 A. The structure has been solved by molecular replacement. Refinement of the structure by simulated annealing and conventional restrained least-squares yielded for 8.0 to 2.3 A data a final R-value of 19.1%. Differences to the known structures of porcine PSTI complexed with trypsinogen and modified human PSTI complexed with chymotrypsinogen occur at the flexible N-terminal part of the molecule. These differences are influenced by crystal packing, as are low temperature factors for the binding loop. The geometry of the binding loop is similar to the complexed structures.

Recombinant human retinoic acid receptor alpha. Binding of DNA and synthetic retinoids to the protein expressed in Escherichia coli.

The human retinoic acid receptor alpha was expressed in Escherichia coli. The recombinant protein was found to be very unstable in several E. coli strains, probably due to proteolysis. Conditions were established to obtain reasonable amounts of active protein for ligand and DNA binding studies. The recombinant receptor showed the expected DNA binding activities in gel-retardation assays. Ligand binding properties were measured in a charcoal absorption assay. The dissociation constant for highly specific bound retinoic acid was found to be 0.67 nM. The affinity of several synthetic retinoids to the recombinant protein was determined and compared to their biological activity. Some of the values presented here differ significantly from those published earlier for the receptor or its isolated hormone-binding domain.

Three-dimensional structure of the complexes between bovine chymotrypsinogen A and two recombinant variants of human pancreatic secretory trypsin inhibitor (Kazal-type).

Variants of the human pancreatic secretory trypsin inhibitor (PSTI) have been created during a protein design project to generate a high-affinity inhibitor with respect to some serine proteases other than trypsin. Two modified versions of human PSTI with high affinity for chymotrypsin were crystallized as a complex with chymotrypsinogen. Both crystallize isomorphously in space group P4(1)2(1)2 with lattice constants a = 84.4 A, c = 86.7 A and diffract to 2.3 A resolution. The structure was solved by molecular replacement. The final R-value after refinement with 8.0 to 2.3 A resolution data was 19.5% for both complexes after inclusion of about 50 bound water molecules. The overall three-dimensional structure of PSTI is similar to the structure of porcine PSTI in the trypsinogen complex (1TGS). Small differences in the relative orientation of the binding loop and the core of the inhibitors indicate flexible adaptation to the proteases. The chymotrypsinogen part of the complex is similar to chymotrypsin. After refolding induced by binding of the inhibitor the root-mean-square difference of the active site residues A186 to A195 and A217 to A222 compared to chymotrypsin was 0.26 A.

A phasmid optimised for protein design projects: pMAMPF.

Protein design requires the rapid production of recombinant genes and active recombinant proteins, the latter in sufficient amounts for functional and physical studies. We present here the construction and application of a new phasmid vector system, using the fd phage origin, lambda pL promoter, ompA-leader sequence and pMB1 origin, which allows the preparation of secretable proteins in active form, mutagenesis and gene sequencing, without subcloning steps. The vector can be used in plasmid form in a stably transformed culture to induce product formation, or as a packaged single-stranded phasmid, which, via batch transduction in a growing culture, leads directly to recombinant protein formation. This latter method has the advantage that, during the short period required for phasmid amplification, little counterselection against clones with high rDNA-protein synthesis potential occurs. The total sequence of pMAMPF-1 and pMAMPF-3 can be assembled from known sequences of constituent fragments. Mutated regions were directly sequenced.

Human leukocyte elastase inhibitors: designed variants of human pancreatic secretory trypsin inhibitor (hPSTI).

Variants of human secretory trypsin inhibitor were constructed with the aim of producing inhibitors specific for human leukocyte elastase. Models of the hPSTI/HLE and hPSTI/chymotrypsin complexes were generated by computer aided protein design and used to plan better HLE inhibitors. This resulted in the production of the strongest and most specific inhibitors of HLE known.