a b s t r a c tNaturally 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.

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 exteinthat 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 fromPyrococcus horikoshii. 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 the less robust splicing variants by eliminating the unfavorable extein-intein interactions observed in the structure. This work provides an example of how unveiling structure-function relationships of inteins offers a promising way of improving their properties as better tools for protein engineering.

Protein splicing is an autocatalytic process involving self-excision of an internal protein domain, the intein, and concomitant ligation of the two flanking sequences, the exteins, with a peptide bond. Protein splicing can also take place in trans by naturally split inteins or artificially split inteins, ligating the exteins on two different polypeptide chains into one polypeptide chain. Protein trans-splicing could work in foreign contexts by replacing the native extein sequences with other protein sequences. Protein ligation using protein trans-splicing increasingly becomes a useful tool for biotechnological applications such as semi-synthesis of proteins, segmental isotopic labeling, and in vivo protein engineering. However, only a few split inteins have been successfully applied for protein ligation. Naturally split inteins have been widely used, but they are cross-reactive to each other, limiting their applications to multiple-fragment ligation. Based on the three-dimensional structures including two newly determined intein structures, we derived 21 new split inteins from four highly efficient cis-splicing inteins, in order to develop novel split inteins suitable for protein ligation. We systematically compared trans-splicing of 24 split inteins and tested the cross-activities among them to identify orthogonal split intein fragments that could be used in chemical biology and biotechnological applications.

Background: Borrelia burgdorferi OspE protein recruits complement regulator FH onto the bacteria for immune evasion. Results: We solved the structure of OspE and the OspE⅐FH complex by NMR and x-ray crystallography. Conclusion: The OspE⅐FH 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.

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Inteins are mobile genetic elements that are spliced out of proteins after translation. Some inteins contain a homing endonuclease (HEN) responsible for their propagation. Hedgehog/INTein (HINT) domains catalyzing protein splicing and their nested HEN domains are thought to be functionally independent because of the existence of functional mini-inteins without HEN domains. Despite the lack of obvious mutualism between HEN and HINT domains, HEN domains are persistently found at one specific site in inteins, indicating their potential functional role in protein splicing. Here we report crystal structures of inactive and active mini-inteins derived from inteins residing in the transcription factor IIB of Methanococcus jannaschii and Methanocaldococcus vulcanius, revealing a novel modified HINT fold that might provide new insights into the mutualism between the HEN and HINT domains. We propose an evolutionary model of inteins and a functional role of HEN domains in inteins.

Background: Antimicrobial peptides are new antibiotics avoiding resistance problems. Results: Eurocin is a new antimicrobial peptide featuring a cysteine-stabilized ␣␤-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.

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 transesterification 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. DatabaseThe resonance assignments and structure coordinates have been deposited in BMRB (18653) and RCSB (2LWY)