Jesper S. Oeemig

Solution structure of DnaE intein from Nostoc punctiforme: Structural basis for the design of a new split intein suitable for site‐specific chemical modification

Naturally split DnaE intein from Nostoc punctiforme (Npu) has robust protein trans‐splicing activity and high tolerance of sequence variations at the splicing junctions. We determined the solution structure of a single chain variant of NpuDnaE intein by NMR spectroscopy. Based on the NMR structure and the backbone dynamics of the single chain NpuDnaE intein, we designed a functional split variant of the NpuDnaE intein having a short C‐terminal half (C‐intein) composed of six residues. In vivo and in vitro protein ligation of model proteins by the newly designed split intein were demonstrated.

Eurocin, a New Fungal Defensin: STRUCTURE, LIPID BINDING, AND ITS MODE OF ACTION*

Background: Antimicrobial peptides are new antibiotics avoiding resistance problems. Results: Eurocin is a new antimicrobial peptide featuring a cysteine-stabilized αβ-fold. Eurocin binds the cell wall precursor lipid II but does not disrupt cell membranes. Conclusion: Eurocin acts by inhibiting cell wall synthesis. Its structure is typical for invertebrate defensins. Significance: Knowing the mode of action and structure is a prerequisite for pharmaceutical application of an antibiotic. Antimicrobial peptides are a new class of antibiotics that are promising for pharmaceutical applications because they have retained efficacy throughout evolution. One class of antimicrobial peptides are the defensins, which have been found in different species. Here we describe a new fungal defensin, eurocin. Eurocin acts against a range of Gram-positive human pathogens but not against Gram-negative bacteria. Eurocin consists of 42 amino acids, forming a cysteine-stabilized α/β-fold. The thermal denaturation data point shows the disulfide bridges being responsible for the stability of the fold. Eurocin does not form pores in cell membranes at physiologically relevant concentrations; it does, however, lead to limited leakage of a fluorophore from small unilamellar vesicles. Eurocin interacts with detergent micelles, and it inhibits the synthesis of cell walls by binding equimolarly to the cell wall precursor lipid II.

NMR and Crystal Structures of the Pyrococcus horikoshii RadA Intein Guide a Strategy for Engineering a Highly Efficient and Promiscuous Intein.

In protein splicing, an intervening protein sequence (intein) in the host protein excises itself out and ligates two split host protein sequences (exteins) to produce a mature host protein. Inteins require the involvement for the splicing of the first residue of the extein that follows the intein (which is Cys, Ser, or Thr). Other extein residues near the splicing junctions could modulate splicing efficiency even when they are not directly involved in catalysis. Mutual interdependence between this molecular parasite (intein) and its host protein (exteins) is not beneficial for intein spread but could be advantageous for intein survival during evolution. Elucidating extein-intein dependency has increasingly become important since inteins are recognized as useful biotechnological tools for protein ligation. We determined the structures of one of inteins with high splicing efficiency, the RadA intein from Pyrococcus horikoshii (PhoRadA). The solution NMR structure and the crystal structures elucidated the structural basis for its high efficiency and directed our efforts of engineering that led to rational design of a functional minimized RadA intein. The crystal structure of the minimized RadA intein also revealed the precise interactions between N-extein and the intein. We systematically analyzed the effects at the -1 position of N-extein and were able to significantly improve the splicing efficiency of a less robust splicing variant by eliminating the unfavorable extein-intein interactions observed in the structure. This work provides an example of how unveiling structure-function relationships of inteins offer a promising way of improving their properties as better tools for protein engineering.

Structural basis for complement evasion by Lyme disease pathogen Borrelia burgdorferi

Background: Borrelia burgdorferi OspE protein recruits complement regulator FH onto the bacteria for immune evasion. Results: We solved the structure of OspE and the OspE·FH complex by NMR and x-ray crystallography. Conclusion: The OspE·FH structure shows how Borrelia evade complement attack by mimicking how host cells protect themselves. Significance: This explains how the bacteria survive in the host and facilitates vaccine design against borreliosis. Borrelia burgdorferi spirochetes that cause Lyme borreliosis survive for a long time in human serum because they successfully evade the complement system, an important arm of innate immunity. The outer surface protein E (OspE) of B. burgdorferi is needed for this because it recruits complement regulator factor H (FH) onto the bacterial surface to evade complement-mediated cell lysis. To understand this process at the molecular level, we used a structural approach. First, we solved the solution structure of OspE by NMR, revealing a fold that has not been seen before in proteins involved in complement regulation. Next, we solved the x-ray structure of the complex between OspE and the FH C-terminal domains 19 and 20 (FH19-20) at 2.83 Å resolution. The structure shows that OspE binds FH19-20 in a way similar to, but not identical with, that used by endothelial cells to bind FH via glycosaminoglycans. The observed interaction of OspE with FH19-20 allows the full function of FH in down-regulation of complement activation on the bacteria. This reveals the molecular basis for how B. burgdorferi evades innate immunity and suggests how OspE could be used as a potential vaccine antigen.

Intermolecular domain swapping induces intein-mediated protein alternative splicing.

Protein sequences are diversified on the DNA level by recombination and mutation and can be further increased on the RNA level by alternative RNA splicing, involving introns that have important roles in many biological processes. The protein version of introns (inteins), which catalyze protein splicing, were first reported in the 1990s. The biological roles of protein splicing still remain elusive because inteins neither provide any clear benefits nor have an essential role in their host organisms. We now report protein alternative splicing, in which new protein sequences can be produced by protein recombination by intermolecular domain swapping of inteins, as elucidated by NMR spectroscopy and crystal structures. We demonstrate that intein-mediated protein alternative splicing could be a new strategy to increase protein diversity (that is, functions) without any modification in genetic backgrounds. We also exploited it as a post-translational protein conformation-driven switch of protein functions (for example, as highly specific protein interference).

Structural basis for protein trans‐splicing by a bacterial intein‐like domain – protein ligation without nucleophilic side chains

Protein splicing in trans by split inteins has become a useful tool for protein engineering in vivo and in vitro. Inteins require Cys, Ser or Thr at the first residue of the C‐terminal flanking sequence because a thiol or hydroxyl group in the side chains is a nucleophile indispensable for the trans‐esterification step during protein splicing. Newly‐identified distinct sequences with homology to the hedgehog/intein superfamily, called bacterial intein‐like (BIL) domains, often do not have Cys, Ser, or Thr as the obligatory nucleophilic residue found in inteins. We demonstrated that BIL domains from Clostridium thermocellum (Cth) are proficient at protein splicing without any sequence changes. We determined the first solution NMR structure of a BIL domain, CthBIL4, to guide engineering of split BIL domains for protein ligation. The newly‐engineered split BIL domain could catalyze protein ligation by trans‐splicing. Protein ligation without any nucleophilic residues of Cys, Ser and Thr could alleviate junction sequence requirements for protein trans‐splicing imposed by split inteins and could broaden applications of protein ligation by protein trans‐splicing.

NMR resonance assignment of DnaE intein from Nostoc punctiforme

DnaE intein from Nostoc punctiforme (Npu) is one of naturally occurring split inteins, which has robust protein splicing activity. Highly efficient trans-splicing activity of NpuDnaE intein could widen various biotechnological applications. However, structural basis of the efficient protein splicing activity is poorly understood. As a first step toward better understanding of protein trans-splicing mechanism, we present the backbone and side-chain resonance assignments of a single chain variant NpuDnaE intein as determined by triple resonance experiments with [13C,15N]-labeled protein.

A monomeric TIM-barrel structure from Pyrococcus furiosus is optimized for extreme temperatures.

The structure of phosphoribosyl anthranilate isomerase (TrpF) from the hyperthermophilic archaeon Pyrococcus furiosus (PfTrpF) has been determined at 1.75 Å resolution. The PfTrpF structure has a monomeric TIM-barrel fold which differs from the dimeric structures of two other known thermophilic TrpF proteins. A comparison of the PfTrpF structure with the two known bacterial thermophilic TrpF structures and the structure of a related mesophilic protein from Escherichia coli (EcTrpF) is presented. The thermophilic TrpF structures contain a higher proportion of ion pairs and charged residues compared with the mesophilic EcTrpF. These residues contribute to the closure of the central barrel and the stabilization of the barrel and the surrounding α-helices. In the monomeric PfTrpF conserved structural water molecules are mostly absent; instead, the structural waters are replaced by direct side-chain-main-chain interactions. As a consequence of these combined mechanisms, the P. furiosus enzyme is a thermodynamically stable and entropically optimized monomeric TIM-barrel enzyme which defines a good framework for further protein engineering for industrial applications.

Cloning, expression, purification, crystallization and preliminary X-ray diffraction data of the Pyrococcus horikoshii RadA intein

The RadA intein from the hyperthermophilic archaebacterium Pyrococcus horikoshii was cloned, expressed and purified for subsequent structure determination. The protein crystallized rapidly in several conditions. The best crystals, which diffracted to 1.75 Å resolution, were harvested from drops consisting of 0.1 M HEPES pH 7.5, 3.0 M NaCl and were cryoprotected with Paratone-N before flash-cooling. The collected data were processed in the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 58.1, b = 67.4, c = 82.9 Å. Molecular replacement with Rosetta using energy- and density-guided structure optimization provided the initial solution, which is currently under refinement.

Backbone and sidechain 1H, 13C and 15N resonance assignments of the human brain-type fatty acid binding protein (FABP7) in its apo form and the holo forms binding to DHA, oleic acid, linoleic acid and elaidic acid.

In this manuscript, we present the backbone and side chain assignments of human brain-type fatty acid binding protein, also known as FABP7, in its apo form and in four different holo forms, bound to DHA, oleic acid, linoleic acid and elaidic acid.