LAGLIDADG homing endonucleases (LHEs) are a family of highly specific DNA endonucleases capable of recognizing target sequences ∼20 bp in length, thus drawing intense interest for their potential academic, biotechnological and clinical applications. Methods for rational design of LHEs to cleave desired target sites are presently limited by a small number of high-quality native LHEs to serve as scaffolds for protein engineering-many are unsatisfactory for gene targeting applications. One strategy to address such limitations is to identify close homologs of existing LHEs possessing superior biophysical or catalytic properties. To test this concept, we searched public sequence databases to identify putative LHE open reading frames homologous to the LHE I-AniI and used a DNA binding and cleavage assay using yeast surface display to rapidly survey a subset of the predicted proteins. These proteins exhibited a range of capacities for surface expression and also displayed locally altered binding and cleavage specificities with a range of in vivo cleavage activities. Of these enzymes, I-HjeMI demonstrated the greatest activity in vivo and was readily crystallizable, allowing a comparative structural analysis. Taken together, our results suggest that even highly homologous LHEs offer a readily accessible resource of related scaffolds that display diverse biochemical properties for biotechnological applications.

Computational enzyme design holds promise for the production of renewable fuels, drugs and chemicals. De novo enzyme design has generated catalysts for several reactions, but with lower catalytic efficiencies than naturally occurring enzymes. Here we report the use of game-driven crowdsourcing to enhance the activity of a computationally designed enzyme through the functional remodeling of its structure. Players of the online game Foldit were challenged to remodel the backbone of a computationally designed bimolecular Diels-Alderase to enable additional interactions with substrates. Several iterations of design and characterization generated a 24-residue helix-turn-helix motif, including a 13-residue insertion, that increased enzyme activity >18-fold. X-ray crystallography showed that the large insertion adopts a helix-turn-helix structure positioned as in the Foldit model. These results demonstrate that human creativity can extend beyond the macroscopic challenges encountered in everyday life to molecular-scale design problems.

A type IIG restriction endonuclease, RM.BpuSI from Bacillus pumilus, has been characterized and its X-ray crystal structure determined at 2.35Å resolution. The enzyme is comprised of an array of 5-folded domains that couple the enzyme's N-terminal endonuclease domain to its C-terminal target recognition and methylation activities. The REase domain contains a PD-x(15)-ExK motif, is closely superimposable against the FokI endonuclease domain, and coordinates a single metal ion. A helical bundle domain connects the endonuclease and methyltransferase (MTase) domains. The MTase domain is similar to the N6-adenine MTase M.TaqI, while the target recognition domain (TRD or specificity domain) resembles a truncated S subunit of Type I R-M system. A final structural domain, that may form additional DNA contacts, interrupts the TRD. DNA binding and cleavage must involve large movements of the endonuclease and TRD domains, that are probably tightly coordinated and coupled to target site methylation status.

OBJECTIVE To determine the effectiveness and safety of short-term treatment of infliximab (IFX) in a group of Chinese patients with active Crohn's disease (CD). METHODS Patients with established diagnosis of active CD were treated with IFX intravenously with a dose of 5 mg/kg at week 0, 2, 6. Clinical assessments were performed at baseline (week 0) and every week after IFX infusion until 8 weeks after the induction dose. RESULTS Fourteen patients (nine male, five female) with a mean age of 29.7 years (range from 15 to 65 years) were included in the analysis. The mean subjective scores were decreased from 2.85 ± 0.57 at baseline to 1.3 ± 0.4 at week 14 (P < 0.05). The mean Harvey-Bradshaw index was 7.9 ± 1.5 at baseline and 2.3 ± 1.0 at week 14. The levels of erythrocyte sedimentation rate, serum C-reactive protein, total protein (TP) and albumin (ALB) were significantly improved during the 14-week period. Colonoscopy showed a remarkable improvement. Mild and transient adverse events including skin itching, headache and elevation of serum alanine aminotransferase and aspartate transaminase were each observed in one patient. Severe anemia, leucopenia and thrombocytopenia at week 27 after three infusions of IFX were observed in one patient. CONCLUSIONS Treatment with three infusions of IFX at a dose of 5 mg/kg was effective for induction of remission for active CD patients who failed to respond to conventional therapies. Study of long-term efficacy and safety of IFX therapy is warranted for further investigations.

The crystal structure of the rare-cutting HNH restriction endonuclease PacI in complex with its eight-base-pair target recognition sequence 5'-TTAATTAA-3' has been determined to 1.9 A resolution. The enzyme forms an extended homodimer, with each subunit containing two zinc-bound motifs surrounding a betabetaalpha-metal catalytic site. The latter is unusual in that a tyrosine residue likely initiates strand cleavage. PacI dramatically distorts its target sequence from Watson-Crick duplex DNA base pairing, with every base separated from its original partner. Two bases on each strand are unpaired, four are engaged in noncanonical A:A and T:T base pairs, and the remaining two bases are matched with new Watson-Crick partners. This represents a highly unusual DNA binding mechanism for a restriction endonuclease, and implies that initial recognition of the target site might involve significantly different contacts from those visualized in the DNA-bound cocrystal structures.

OBJECTIVE: To investigated the potential and safety of the monoclonal antibody to TNFalpha infliximab (IFX) in the treatment of active Crohn's disease (CD). METHODS: Patients who were confirmed diagnosis of CD and were unresponsive to the conventional treatments, or recurred after surgeries, or discontinued treatment due to drug intolerance, were treated with IFX intravenously in a dose of 5 mg/kg at week 0, 2, 6 (IFX infusion continued at an interval of every 8 weeks if respond to initial dosing). Clinical assessments, including disease activity, blood biological markers and colonoscopic findings, were performed at baseline (week 0) and each week (4 weeks or later for colonoscopy) after IFX infusion were conducted until the week before 4(th) infusion from initiated. RESULTS: Ten patients (8 male, 2 female) with mean age of 31.4 years (ranged from 15 to 65 years old) were included in the analysis. The mean subjective score from baseline to week 14 was decreased from 2.2 +/- 0.6 to 1.2 +/- 0.4 (P < 0.05). The mean Harvey-Bradshaw index was 6.6 +/- 1.6 at baseline and 2.1 +/- 1.0 at week 14. The levels of ESR, CRP, serum total protein (TP) and albumin (Alb) were significantly improved during the 14-week period. Colonoscopy showed a remarkable improvement of Crohn's Disease Endoscopic Index of Severity (CDEIS). No infusion-related reaction was observed in all patients during the treatment. Mild or transient skin itching and headache were respectively reported in two patients. Transient elevation of serum ALT and AST after 3(rd) infusion in one patient, and severe anemia including leucopenia and thrombocytopenia at week 35 after 1(st) infusion in one male patient were observed. CONCLUSIONS: Treatment with three infusions of IFX in a dose of 5 mg/kg was effective for induction of remission for active and complex CD patients who failed to respond to conventional treatment. Long-term safety of the therapy effect was warranted in further investigations.

Proteins of the DUF199 family, present in all Gram-positive bacteria and best characterized by the WhiA sporulation control factor in Streptomyces coelicolor, are thought to act as genetic regulators. The crystal structure of the DUF199/WhiA protein from Thermatoga maritima demonstrates that these proteins possess a bipartite structure, in which a degenerate N-terminal LAGLIDADG homing endonuclease (LHE) scaffold is tethered to a C-terminal helix-turn-helix (HTH) domain. The LHE domain has lost those residues critical for metal binding and catalysis, and also displays an extensively altered DNA-binding surface as compared with homing endonucleases. The HTH domain most closely resembles related regions of several bacterial sigma70 factors that bind the -35 regions of bacterial promoters. The structure illustrates how an invasive element might be transformed during evolution into a larger assemblage of protein folds that can participate in the regulation of a complex biological pathway.

Factor VIII (fVIII) is a serum protein in the coagulation cascade that nucleates the assembly of a membrane-bound protease complex on the surface of activated platelets at the site of a vascular injury. Hemophilia A is caused by a variety of mutations in the factor VIII gene and typically requires replacement therapy with purified protein. We have determined the structure of a fully active, recombinant form of factor VIII (r-fVIII), which consists of a heterodimer of peptides, respectively containing the A1-A2 and A3-C1-C2 domains. The structure permits unambiguous modeling of the relative orientations of the five domains of r-fVIII. Comparison of the structures of factor VIII, factor V and ceruloplasmin indicates that the location of bound metal ions and of glycosylation, both of which are critical for domain stabilization and association, overlap at some positions but have diverged at others.

The thermodynamic profiles of target site recognition have been surveyed for homing endonucleases from various structural families. Similar to DNA-binding proteins that recognize shorter target sites, homing endonucleases display a narrow range of binding free energies and affinities, mediated by structural interactions that balance the magnitude of enthalpic and entropic forces. While the balance of DeltaH and TDeltaS are not strongly correlated with the overall extent of DNA bending, unfavorable DeltaH(binding) is associated with unstacking of individual base steps in the target site. The effects of deleterious basepair substitutions in the optimal target sites of two LAGLIDADG homing endonucleases, and the subsequent effect of redesigning one of those endonucleases to accommodate that DNA sequence change, were also measured. The substitution of base-specific hydrogen bonds in a wild-type endonuclease/DNA complex with hydrophobic van der Waals contacts in a redesigned complex reduced the ability to discriminate between sites, due to nonspecific DeltaS(binding).

I-HmuI and I-BasI are two highly similar nicking DNA endonucleases, which are each encoded by a group I intron inserted into homologous sites within the DNA polymerase genes of Bacillus phages SPO1 and Bastille, respectively. Here, we present a comparison of the DNA specificities and cleavage activities of these enconucleases with homologous target sites. I-BasI has properties that are typical of homing endonucleases, nicking the intron-minus polymerase genes in either host genome, three nucleotides downstream of the intron insertion site. In contrast, I-HmuI nicks both the intron-plus and intron-minus site in its own host genome, but does not act on the target from Bastille phage. Although the enzymes have distinct DNA substrate specificities, both bind to an identical 25bp region of their respective intron-minus DNA polymerase genes surrounding the intron insertion site. The endonucleases appear to interact with the DNA substrates in the downstream exon 2 in a similar manner. However, whereas I-HmuI is known to make its only base-specific contacts within this exon region, structural modeling analyses predict that I-BasI might make specific base contacts both upstream and downstream of the site of intron insertion. The predicted requirement for base-specific contacts in exon 1 for cleavage by I-BasI was confirmed experimentally. This explains the difference in substrate specificities between the two enzymes, including the observation that the former enzyme is relatively insensitive to the presence of an intron upstream of exon 2. These differences are likely a consequence of divergent evolutionary constraints.