An Intrinsically Disordered Peptide-Peptide Stapler for Highly Efficient Protein Ligation Both in Vivo and in Vitro.

Herein, we report an intrinsically disordered protein SpyStapler that can catalyze the isopeptide bond formation between two peptide tags, that is, SpyTag and BDTag, both in vitro and in vivo. SpyStapler and BDTag are developed by splitting SpyCatcher-the cognate protein partner of SpyTag-at the more solvent exposed second loop region. Regardless of their locations in protein constructs, SpyStapler enables efficient covalent coupling of SpyTag and BDTag under a variety of mild conditions in vitro (yield ∼80%). Co-expression of SpyStapler with telechelic dihydrofolate reductase (DHFR) bearing a SpyTag at N-terminus and a BDTag at C-terminus leads to direct cellular synthesis of a circular DHFR. Mechanistic studies involving circular dichroism and nuclear magnetic resonance spectrometry reveal that SpyStapler alone is disordered in solution and forms a stable folded structure ( Tm ∼ 55 °C) in the presence of both SpyTag and BDTag upon isopeptide bonding. No ordered structure can be formed in the absence of either tag. The catalytically inactive SpyStapler-EQ mutant cannot form a stable physical complex with SpyTag and BDTag, but it can fold into ordered structure in the presence of the ligated product (SpyTag-BDTag). It suggests that the isopeptide bond is important in stabilizing the complex. Given its efficiency, resilience, and robustness, SpyStapler provides new opportunities for bioconjugation and creation of complex protein architectures.

Self-replication reactions dependent on tertiary interaction motifs in an RNA ligase ribozyme.

RNA can function both as an informational molecule and as a catalyst in living organisms. This duality is the premise of the RNA world hypothesis. However, one flaw in the hypothesis that RNA was the most essential molecule in primitive life is that no RNA self-replicating system has been found in nature. To verify whether RNA has the potential for self-replication, we constructed a new RNA self-assembling ribozyme that could have conducted an evolvable RNA self-replication reaction. The artificially designed, in vitro selected ligase ribozyme was employed as a prototype for a self-assembling ribozyme. The ribozyme is composed of two RNA fragments (form R1·Z1) that recognize another R1·Z1 molecule as their substrate and perform the high turnover ligation reaction via two RNA tertiary interaction motifs. Furthermore, the substrate recognition of R1·Z1 is tolerant of mutations, generating diversity in the corresponding RNA self-replicating network. Thus, we propose that our system implies the significance of RNA tertiary motifs in the early RNA molecular evolution of the RNA world.

In vitro selection of ribozymes dependent on peptides for activity.

A peptide-dependent ribozyme ligase (aptazyme ligase) has been selected from a random sequence population based on the small L1 ligase. The aptazyme ligase is activated > 18,000-fold by its cognate peptide effector, the HIV-1 Rev arginine-rich motif (ARM), and specifically recognizes the Rev ARM relative to other peptides containing arginine-rich motifs. Moreover, the aptazyme ligase can preferentially recognize the Rev ARM in the context of the full-length HIV-1 Rev protein. The only cross-reactivity exhibited by the aptazyme is toward the Tat ARM. Reselection of peptide- and protein-dependent aptazymes from a partially randomized population yielded aptazymes that could readily discriminate against the Tat ARM. These results have important implications for the development of aptazymes that can be used in arrays for the detection and quantitation of multiple cellular proteins (proteome arrays).

A de novo designed peptide ligase: a mechanistic investigation.

A 33-residue de novo designed peptide ligase is reported which catalyzes the template-directed condensation of suitably activated short peptides with catalytic efficiencies in excess of 10(5) ([k(cat)/K(m)]/k(uncat)). The ligase peptide, derived from natural and designed alpha-helical coiled-coil proteins, presents a surface for substrate assembly via formation of a hydrophobic core at the peptide interface. Charged residues flanking the core provide additional binding specificity through electrostatic complementarity. Addition of the template to an equimolar fragment solution results in up to 4100-fold increases in initial reaction rates. Dramatic decreases in efficiency upon mutation of charged residues or increase in ionic strength establishes the importance of electrostatic recognition to ligase efficiency. Although most of the increase in reaction efficiency is due to entropic gain from binding of substrates in close proximity, mechanistic studies with altered substrates demonstrate that the system is highly sensitive to precise ordering at the point of ligation. Taken together these results represent the first example of a peptide catalyst with designed substrate binding sites which can significantly accelerate a bimolecular reaction and support the general viability of alpha-helical protein assemblies in artificial enzyme design.

Design and optimization of effector-activated ribozyme ligases.

A selected ribozyme ligase, L1, has been engineered to respond to small organic effectors. Residues important for ribozyme catalysis were mapped to a compact core structure. Aptamers that bound adenosine and theophylline were appended to the core structure, and the resultant aptazymes proved to be responsive to their cognate effectors. Rational sequence substitutions in the joining region between the aptamer and the ribozyme yielded aptazymes whose activities were enhanced from 800-1600-fold in the presence of 1 mM ATP or theophylline, respectively. However, when an anti-flavin aptamer was appended to the core ribozyme structure flavin-responsivity was minimal. The joining region between the aptamer and the ribozyme core was randomized and a series of negative and positive selection steps yielded aptazymes that were activated by up to 260-fold in the presence of 100 microM FMN. The selected joining regions proved to be 'communication modules' that could be used to join other aptamers to the ribozyme core to form aptazymes. These results show that ribozyme ligases can be readily engineered to function as allosteric enzymes, and reveal that many of the techniques and principles previously demonstrated during the development of hammerhead aptazymes may be generalizable.

A pH-tunable peptide ligase.

A chemical ligation system is reported, in which a highly acidic coiled-coil peptide was used to template two basic peptide fragments and catalyze their condensation, in a pH-tunable fashion, to generate a coiled-coil product. This template showed a high catalytic efficiency (with single turnover) under neutral conditions. Under acidic conditions, however, its catalytic efficiency was reduced by approximately 4500-fold.

A synthetic peptide ligase.

The preparation of synthetic molecules showing the remarkable efficiencies characteristic of natural biopolymer catalysts remains a formidable challenge for chemical biology. Although significant advances have been made in the understanding of protein structure and function, the de novo construction of such systems remains elusive. Re-engineered natural enzymes and catalytic antibodies, possessing tailored binding pockets with appropriately positioned functional groups, have been successful in catalysing a number of chemical transformations, sometimes with impressive efficiencies. But efforts to produce wholly synthetic catalytic peptides have typically resulted in compounds with questionable structural stability, let alone reactivity. Here we describe a 33-residue synthetic peptide, based on the coiled-coil structural motif, which efficiently catalyses the condensation of two shorter peptide fragments with high sequence- and diastereoselectivity. Depending on the substrates used, we observe rate enhancements of tenfold to 4,100-fold over the background, with catalytic efficiencies in excess of 10(4). These results augur well for the rational design of functional peptides.

Several mammalian ubiquitin carrier proteins, but not E2(20K), are related to the 20-kDa yeast E2, RAD6.

Western blot analysis was used to probe the relationships between the multiple ubiquitin carrier proteins (E2 s) of rabbit reticulocytes and the 20-kDa E2 encoded by the RAD6 gene of the yeast S. cerevisiae. Reticulocyte E2-14K, E2-17K, and E2-25K each reacted with two or more polyclonal anti-RAD6 antibody preparations; E2-20K, E2-35K, and E2-230K did not cross-react. These results suggest that some, but not all, reticulocyte E2 s are members of a RAD6-like protein family which is conserved within and across species. RAD6 and E2-20K were also shown to multi-ubiquitinate histones by different mechanisms.