Birte Höcker

Computational design of ligand binding is not a solved problem

Computational design has been very successful in recent years: multiple novel ligand binding proteins as well as enzymes have been reported. We wanted to know in molecular detail how precise the predictions of the interactions of protein and ligands are. Therefore, we performed a structural analysis of a number of published receptors designed onto the periplasmic binding protein scaffold that were reported to bind to the new ligands with nano- to micromolar affinities. It turned out that most of these designed proteins are not suitable for structural studies due to instability and aggregation. However, we were able to solve the crystal structure of an arabinose binding protein designed to bind serotonin to 2.2 Å resolution. While crystallized in the presence of an excess of serotonin, the protein is in an open conformation with no serotonin bound, although the side-chain conformations in the empty binding pocket are very similar to the conformations predicted. During subsequent characterization using isothermal titration calorimetry, CD, and NMR spectroscopy, no indication of binding could be detected for any of the tested designed receptors, whereas wild-type proteins bound their ligands as expected. We conclude that although the computational prediction of side-chain conformations appears to be working, it does not necessarily confer binding as expected. Hence, the computational design of ligand binding is not a solved problem and needs to be revisited.

De novo design of a four-fold symmetric TIM-barrel protein with atomic-level accuracy

Despite efforts for over 25 years, de novo protein design has not succeeded in achieving the TIM-barrel fold. Here we describe the computational design of 4-fold symmetrical (β/α)8-barrels guided by geometrical and chemical principles. Experimental characterization of 33 designs revealed the importance of sidechain-backbone hydrogen bonding for defining the strand register between repeat units. The X-ray crystal structure of a designed thermostable 184-residue protein is nearly identical with the designed TIM-barrel model. PSI-BLAST searches do not identify sequence similarities to known TIM-barrel proteins, and sensitive profile-profile searches indicate that the design sequence is distant from other naturally occurring TIM-barrel superfamilies, suggesting that Nature has only sampled a subset of the sequence space available to the TIM-barrel fold. The ability to de novo design TIM-barrels opens new possibilities for custom-made enzymes.

A beta alpha-barrel built by the combination of fragments from different folds.

Combinatorial assembly of protein domains plays an important role in the evolution of proteins. There is also evidence that protein domains have come together from stable subdomains. This concept of modular assembly could be used to construct new well folded proteins from stable protein fragments. Here, we report the construction of a chimeric protein from parts of a (βα)8-barrel enzyme from histidine biosynthesis pathway (HisF) and a protein of the (βα)5-flavodoxin-like fold (CheY) from Thermotoga maritima that share a high structural similarity. We expected this construct to fold into a full (βα)8-barrel. Our results show that the chimeric protein is a stable monomer that unfolds with high cooperativity. Its three-dimensional structure, which was solved to 3.1 Å resolution by x-ray crystallography, confirms a barrel-like fold in which the overall structures of the parent proteins are highly conserved. The structure further reveals a ninth strand in the barrel, which is formed by residues from the HisF C terminus and an attached tag. This strand invades between β-strand 1 and 2 of the CheY part closing a gap in the structure that might be due to a suboptimal fit between the fragments. Thus, by a combination of parts from two different folds and a small arbitrary fragment, we created a well folded and stable protein.

Automated scaffold selection for enzyme design

A major goal of computational protein design is the construction of novel functions on existing protein scaffolds. There the first question is which scaffold is suitable for a specific reaction. Given a set of catalytic residues and their spatial arrangement, one wants to identify a protein scaffold that can host this active site. Here, we present an algorithm called ScaffoldSelection that is able to rapidly search large sets of protein structures for potential attachment sites of an enzymatic motif. The method consists of two steps; it first identifies pairs of backbone positions in pocket‐like regions. Then, it combines these to complete attachment sites using a graph theoretical approach. Identified matches are assessed for their ability to accommodate the substrate or transition state. A representative set of structures from the Protein Data Bank (∼3500) was searched for backbone geometries that support the catalytic residues for 12 chemical reactions. Recapitulation of native active site geometries is used as a benchmark for the performance of the program. The native motif is identified in all 12 test cases, ranking it in the top percentile in 5 out of 12. The algorithm is fast and efficient, although dependent on the complexity of the motif. Comparisons to other methods show that ScaffoldSelection performs equally well in terms of accuracy and far better in terms of speed. Thus, ScaffoldSelection will aid future computational protein design experiments by preselecting protein scaffolds that are suitable for a specific reaction type and the introduction of a predefined amino acid motif. Proteins 2009.

Evolutionary relationship of two ancient protein superfolds

Proteins are the molecular machines of the cell that fold into specific three-dimensional structures to fulfill their functions. To improve our understanding of how the structure and function of proteins arises, it is crucial to understand how evolution has generated the structural diversity we observe today. Classically, proteins that adopt different folds are considered to be nonhomologous. However, using state-of-the-art tools for homology detection, we found evidence of homology between proteins of two ancient and highly populated protein folds, the (βα)8-barrel and the flavodoxin-like fold. We detected a family of sequences that show intermediate features between both folds and determined what is to our knowledge the first representative crystal structure of one of its members, giving new insights into the evolutionary link of two of the earliest folds. Our findings contribute to an emergent vision where protein superfolds share common ancestry and encourage further approaches to complete the mapping of structure space onto sequence space.

Binding Pocket Optimization by Computational Protein Design

Engineering specific interactions between proteins and small molecules is extremely useful for biological studies, as these interactions are essential for molecular recognition. Furthermore, many biotechnological applications are made possible by such an engineering approach, ranging from biosensors to the design of custom enzyme catalysts. Here, we present a novel method for the computational design of protein-small ligand binding named PocketOptimizer. The program can be used to modify protein binding pocket residues to improve or establish binding of a small molecule. It is a modular pipeline based on a number of customizable molecular modeling tools to predict mutations that alter the affinity of a target protein to its ligand. At its heart it uses a receptor-ligand scoring function to estimate the binding free energy between protein and ligand. We compiled a benchmark set that we used to systematically assess the performance of our method. It consists of proteins for which mutational variants with different binding affinities for their ligands and experimentally determined structures exist. Within this test set PocketOptimizer correctly predicts the mutant with the higher affinity in about 69% of the cases. A detailed analysis of the results reveals that the strengths of PocketOptimizer lie in the correct introduction of stabilizing hydrogen bonds to the ligand, as well as in the improved geometric complemetarity between ligand and binding pocket. Apart from the novel method for binding pocket design we also introduce a much needed benchmark data set for the comparison of affinities of mutant binding pockets, and that we use to asses programs for in silico design of ligand binding.

A highly stable protein chimera built from fragments of different folds

Proteins increased in complexity during the course of evolution. Domains as well as subdomain-sized fragments were recruited and adapted to form new proteins and novel folds. This concept can be used in engineering to construct new proteins. We previously reported the combination of fragments from two ancient protein folds, a flavodoxin-like and a (βα)₈-barrel protein. Here we report two further attempts at engineering a chimeric protein from fragments of these folds. While one of the constructs showed a high tendency to aggregate, the other turned out to be a highly stable, well-structured protein. In terms of stability against heat and chemical denaturation this chimera, named NarLHisF, is superior to the earlier presented CheYHisF. This is the second instance of a chimera build from two different protein folds, which demonstrates how easily recombination can lead to the development and diversification of new proteins--a mechanism that most likely occurred frequently in the course of evolution. Based on the results of the failed and the successful chimera, we discuss important considerations for a general design strategy for fold chimeras.

Design of proteins from smaller fragments — learning from evolution

Nature has generated an impressive set of proteins with diverse folds and functions. It has been able to do so using mechanisms such as duplication and fusion as well as recombination of smaller protein fragments that serve as building blocks. These evolutionary mechanisms provide a template for the rational design of new proteins from fragments of existing proteins. Design by duplication and fusion has been explored for a number of symmetric protein folds, while design by rational recombination has just emerged. First experiments in recombining fragments from the same and different folds are proving successful in building new proteins that harbor easily evolvable properties originating from the parents. Overall, duplication and recombination of smaller fragments shows much potential for future applications in the design of proteins.

Evolutionary mechanism as a template for protein engineering.

The goal of a protein engineer is to adjust a protein to a specified new function. This is exactly what natural evolution has achieved many times. By studying evolutionary mechanisms, we can learn about ways to use the adaptability of proteins and to build new proteins. In fact, many techniques used in engineering are successfully mimicking evolutionary processes. We introduce the fundamental evolutionary mechanisms, take a closer look at duplication and fusion, recombination, and circular permutation and discuss their influence on protein engineering. Some important techniques are presented and illustrated with examples. Copyright

Engineering the Enolase Magnesium II Binding Site -Implications for its Evolution.

The glycolytic enzyme enolase catalyzes the reversible elimination of water from 2-phosphoglycerate (2-PGA) to form phosphoenolpyruvate (PEP). Two magnesium ions in the active site are thought to facilitate the reaction by activation of the C2 proton of 2-PGA and charge stabilization of the intermediate. The initial abstraction of a proton from a carboxylic acid is common to all members of the enolase superfamily, yet in all other known members of this superfamily, only one magnesium ion (MgI) per active site is sufficient to promote catalysis. We wanted to further investigate the importance of the second magnesium ion (MgII) for the catalytic mechanism of yeast enolase 1. Toward this end, we removed all MgII coordinating residues and replaced substrate-MgII interactions by introducing positively charged side chains. High-resolution crystal structures and activity assays show that the introduced positively charged side chains effectively prohibit MgII binding but fail to promote catalysis. We conclude that enolase is inactive without MgII, yet control mutants without additional positively charged side chains retain basal enolase activity through binding of magnesium to 2-PGA in an open active site without the help of MgII coordinating residues. Thus, we believe that ancestral enolase activity might have evolved in a member of the enolase superfamily that provides only the necessary catalytic residues and the binding site for MgI. Additionally, precatalytic binding of 2-PGA to the apo state of enolase was observed.