Conditional Alternative Protein Splicing Promoted by Inteins from Haloquadratum walsbyi.

Protein splicing is a post-translational process by which an intervening protein, or an intein, catalyzes its own excision from flanking polypeptides, or exteins, coupled to extein ligation. Four inteins interrupt the MCM helicase of the halophile Haloquadratum walsbyi, two of which are mini-inteins that lack a homing endonuclease. Both inteins can be overexpressed in Escherichia coli and purified as unspliced precursors; splicing can be induced in vitro by incubation with salt. However, one intein can splice in 0.5 M NaCl in vitro, whereas the other splices efficiently only in buffer containing over 2 M NaCl; the organism also requires high salt to grow, with the standard growth media containing over 3 M NaCl and about 0.75 M magnesium salts. Consistent with this difference in salt-dependent activity, an intein-containing precursor protein with both inteins promotes conditional alternative protein splicing (CAPS) to yield different spliced products dependent on the salt concentration. Native Trp fluorescence of the inteins suggests that the difference in activity may be due to partial unfolding of the inteins at lower salt concentrations. This differential salt sensitivity of intein activity may provide a useful mechanism for halophiles to respond to environmental changes.

Intein Inhibitors as Novel Antimicrobials: Protein Splicing in Human Pathogens, Screening Methods, and Off-Target Considerations.

Protein splicing is a post-translational process by which an intervening polypeptide, or intein, catalyzes its own removal from the flanking polypeptides, or exteins, concomitant with extein ligation. Although inteins are highly abundant in the microbial world, including within several human pathogens, they are absent in the genomes of metazoans. As protein splicing is required to permit function of essential proteins within pathogens, inteins represent attractive antimicrobial targets. Here we review key proteins interrupted by inteins in pathogenic mycobacteria and fungi, exciting discoveries that provide proof of concept that intein activity can be inhibited and that this inhibition has an effect on the host organism's fitness, and bioanalytical methods that have been used to screen for intein activity. We also consider potential off-target inhibition of hedgehog signaling, given the similarity in structure and function of inteins and hedgehog autoprocessing domains.

An alternative domain-swapped structure of the Pyrococcus horikoshii PolII mini-intein.

Protein splicing is a post-translational process by which an intein catalyzes its own excision from flanking polypeptides, or exteins, concomitant with extein ligation. Many inteins have nested homing endonuclease domains that facilitate their propagation into intein-less alleles, whereas other inteins lack the homing endonuclease (HEN) and are called mini-inteins. The mini-intein that interrupts the DNA PolII of Pyrococcus horikoshii has a linker region in place of the HEN domain that is shorter than the linker in a closely related intein from Pyrococcus abyssi. The P. horikoshii PolII intein requires a higher temperature for catalytic activity and is more stable to digestion by the thermostable protease thermolysin, suggesting that it is more rigid than the P. abyssi intein. We solved a crystal structure of the intein precursor that revealed a domain-swapped dimer. Inteins found as domain swapped dimers have been shown to promote intein-mediated protein alternative splicing, but the solved P. horikoshii PolII intein structure has an active site unlikely to be catalytically competent.

Protein Splicing Activity of the Haloferax volcanii PolB-c Intein Is Sensitive to Homing Endonuclease Domain Mutations.

Inteins are selfish genetic elements residing in open reading frames that can splice post-translationally, resulting in the ligation of an uninterrupted, functional protein. Like other inteins, the DNA polymerase B (PolB) intein of the halophilic archaeon Haloferax volcanii has an active homing endonuclease (HEN) domain, facilitating its horizontal transmission. Previous work has shown that the presence of the PolB intein exerts a significant fitness cost on the organism compared to an intein-free isogenic H. volcanii. Here, we show that mutation of a conserved residue in the HEN domain not only reduces intein homing but also slows growth. Surprisingly, although this mutation is far from the protein splicing active site, it also significantly reduces in vitro protein splicing. Moreover, two additional HEN domain mutations, which could not be introduced to H. volcanii, presumably due to lethality, also eliminate protein splicing activity in vitro. These results suggest an interplay between HEN residues and the protein splicing domain, despite an over 35 Å separation in a PolB intein homology model. The combination of in vivo and in vitro evidence strongly supports a model of codependence between the self-splicing domain and the HEN domain that has been alluded to by previous in vitro studies of protein splicing with HEN domain-containing inteins.

Allosteric Influence of Extremophile Hairpin Motif Mutations on the Protein Splicing Activity of a Hyperthermophilic Intein.

Protein splicing is a post-translational process mediated by an intein, whereby the intein excises itself from a precursor protein with concomitant ligation of the two flanking polypeptides. The intein that interrupts the DNA polymerase II in the extreme hyperthermophile Pyrococcus abyssi has a ß-hairpin that extends the central ß-sheet of the intein. This ß-hairpin is mostly found in inteins from archaea, as well as halophilic eubacteria, and is thus called the extremophile hairpin (EXH) motif. The EXH is stabilized by multiple favorable interactions, including electrostatic interactions involving Glu29, Glu31, and Arg40. Mutations of these residues diminish the extent of N-terminal cleavage and the extent of protein splicing, likely by interfering with the coordination of the steps of splicing. These same mutations decrease the global stability of the intein fold as measured by susceptibility to thermolysin cleavage. 15N-1H heteronuclear single-quantum coherence demonstrated that these mutations altered the chemical environment of active site residues such as His93 (B-block histidine) and Ser166 (F-block residue 4). This work again underscores the connected and coordinated nature of intein conformation and dynamics, where remote mutations can disturb a finely tuned interaction network to inhibit or enhance protein splicing.

Methods to Study the Structure and Catalytic Activity of cis-Splicing Inteins.

The autocatalytic process of protein splicing is facilitated by an intein, which interrupts flanking polypeptides called exteins. The mechanism of protein splicing has been studied by overexpression in E. coli of intein fusion proteins with nonnative exteins. Inteins can be used to generate reactive α-thioesters, as well as proteins with N-terminal Cys residues, to facilitate expressed protein ligation. As such, a more detailed understanding of the function of inteins can have significant impact for biotechnology applications. Here, we provide biochemical methods to study splicing activity and NMR methods to study intein structure and the catalytic mechanism.

Coordination of the third step of protein splicing in two cyanobacterial inteins.

The third step of protein splicing is cyclization of Asn coupled to peptide bond cleavage. In two related cyanobacterial inteins, this step is facilitated by Asn or Gln. For a Synechococcus sp. PCC7002 intein, the isolated third step of protein splicing is more efficient with its native Asn than with substitution to Gln. For a Trichodesmium erythraeum intein, its native Gln facilitates the third step as efficiently as with Asn. Despite these differences, the yield of splicing is not affected, suggesting that the third step is influenced by mechanism-linked conformational changes. A conserved catalytic His and the penultimate residue also play roles in promoting side-chain cyclization.

V67L Mutation Fills an Internal Cavity To Stabilize RecA Mtu Intein.

Inteins mediate protein splicing, which has found extensive applications in protein science and biotechnology. In the Mycobacterium tuberculosis RecA mini-mini intein (ΔΔIhh), a single valine to leucine substitution at position 67 (V67L) dramatically increases intein stability and activity. However, crystal structures show that the V67L mutation causes minimal structural rearrangements, with a root-mean-square deviation of 0.2 Å between ΔΔIhh-V67 and ΔΔIhh-L67. Thus, the structural mechanisms for V67L stabilization and activation remain poorly understood. In this study, we used intrinsic tryptophan fluorescence, high-pressure nuclear magnetic resonance (NMR), and molecular dynamics (MD) simulations to probe the structural basis of V67L stabilization of the intein fold. Guanidine hydrochloride denaturation monitored by fluorescence yielded free energy changes (ΔGf°) of -4.4 and -6.9 kcal mol-1 for ΔΔIhh-V67 and ΔΔIhh-L67, respectively. High-pressure NMR showed that ΔΔIhh-L67 is more resistant to pressure-induced unfolding than ΔΔIhh-V67 is. The change in the volume of folding (ΔVf) was significantly larger for V67 (71 ± 2 mL mol-1) than for L67 (58 ± 3 mL mol-1) inteins. The measured difference in ΔVf (13 ± 3 mL mol-1) roughly corresponds to the volume of the additional methylene group for Leu, supporting the notion that the V67L mutation fills a nearby cavity to enhance intein stability. In addition, we performed MD simulations to show that V67L decreases side chain dynamics and conformational entropy at the active site. It is plausible that changes in cavities in V67L can also mediate allosteric effects to change active site dynamics and enhance intein activity.

Intein-Promoted Cyclization of Aspartic Acid Flanking the Intein Leads to Atypical N-Terminal Cleavage.

Protein splicing is a post-translational reaction facilitated by an intein, or intervening protein, which involves the removal of the intein and the ligation of the flanking polypeptides, or exteins. A DNA polymerase II intein from Pyrococcus abyssi (Pab PolII intein) can promote protein splicing in vitro on incubation at high temperature. Mutation of active site residues Cys1, Gln185, and Cys+1 to Ala results in an inactive intein precursor, which cannot promote the steps of splicing, including cleavage of the peptide bond linking the N-extein and intein (N-terminal cleavage). Surprisingly, coupling the inactivating mutations to a change of the residue at the C-terminus of the N-extein (N-1 residue) from the native Asn to Asp reactivates N-terminal cleavage at pH 5. Similar "aspartic acid effects" have been observed in other proteins and peptides but usually only occur at lower pH values. In this case, however, the unusual N-terminal cleavage is abolished by mutations to catalytic active site residues and unfolding of the intein, indicating that this cleavage effect is mediated by the intein active site and the intein fold. We show via mass spectrometry that the reaction proceeds through cyclization of Asp resulting in anhydride formation coupled to peptide bond cleavage. Our results add to the richness of the understanding of the mechanism of protein splicing and provide insight into the stability of proteins at moderately low pH. The results also explain, and may help practitioners avoid, a side reaction that may complicate intein applications in biotechnology.

Salt-Dependent Conditional Protein Splicing of an Intein from Halobacterium salinarum.

An intein from Halobacterium salinarum can be isolated as an unspliced precursor protein with exogenous exteins after Escherichia coli overexpression. The intein promotes protein splicing and uncoupled N-terminal cleavage in vitro, conditional on incubation with NaCl or KCl at concentrations of >1.5 M. The protein splicing reaction also is conditional on reduction of a disulfide bond between two active site cysteines. Conditional protein splicing under these relatively mild conditions may lead to advances in intein-based biotechnology applications and hints at the possibility that this H. salinarum intein could serve as a switch to control extein activity under physiologically relevant conditions.

Biochemistry in an undergraduate writing-intensive first-year program: Seminar courses in drugs and bioethics.

The College of the Holy Cross offers a universal first-year program called Montserrat, in which first-year students participate in a living-learning experience anchored by a yearlong seminar course. The seminar courses are part of a thematic cluster of four to eight courses; students in the cluster live together in a common dormitory and participate in shared co-curricular events designed to engage the entire cluster in intellectual discourse related to the theme. A two-semester seminar within the "Natural World" cluster was offered using biochemical principles as the underlying content. In the first semester, students were introduced to drug design, activity and abuse via student presentations and guided readings on ethnobotany, drug laws, drug use in religion, and prescription drug costs. In the second semester, students discussed primary readings in ethics followed by case study analyses of assisted reproduction technologies, informed consent, genetic privacy, performance enhancing drugs and genetically modified organisms. Student learning outcomes were evaluated via rubrics and a College-facilitated survey.

Protein splicing: how inteins escape from precursor proteins.

Inteins are nature's escape artists; they facilitate their excision from flanking polypeptides (exteins) concomitant with extein ligation to produce a mature host protein. Splicing requires sequential nucleophilic displacement reactions catalyzed by strategies similar to proteases and asparagine lyases. Inteins require precise reaction coordination rather than rapid turnover or tight substrate binding because they are single turnover enzymes with covalently linked substrates. This has allowed inteins to explore alternative mechanisms with different steps or to use different methods for activation and coordination of the steps. Pressing issues include understanding the underlying details of catalysis and how the splicing steps are controlled.

Recent advances in in vivo applications of intein-mediated protein splicing.

Intein-mediated protein splicing has become an essential tool in modern biotechnology. Fundamental progress in the structure and catalytic strategies of cis- and trans-splicing inteins has led to the development of modified inteins that promote efficient protein purification, ligation, modification and cyclization. Recent work has extended these in vitro applications to the cell or to whole organisms. We review recent advances in intein-mediated protein expression and modification, post-translational processing and labeling, protein regulation by conditional protein splicing, biosensors, and expression of trans-genes.

Internal disulfide bond acts as a switch for intein activity.

Inteins are intervening polypeptides that catalyze their own removal from flanking exteins, concomitant to the ligation of the exteins. The intein that interrupts the DP2 (large) subunit of DNA polymerase II from Methanoculleus marisnigri (Mma) can promote protein splicing. However, protein splicing can be prevented or reduced by overexpression under nonreducing conditions because of the formation of a disulfide bond between two internal intein Cys residues. This redox sensitivity leads to differential activity in different strains of E. coli as well as in different cell compartments. The redox-dependent control of in vivo protein splicing in an intein derived from an anaerobe that can occupy multiple environments hints at a possible physiological role for protein splicing.

Intramolecular disulfide bond between catalytic cysteines in an intein precursor.

Protein splicing is a self-catalyzed and spontaneous post-translational process in which inteins excise themselves out of precursor proteins while the exteins are ligated together. We report the first discovery of an intramolecular disulfide bond between the two active-site cysteines, Cys1 and Cys+1, in an intein precursor composed of the hyperthermophilic Pyrococcus abyssi PolII intein and extein. The existence of this intramolecular disulfide bond is demonstrated by the effect of reducing agents on the precursor, mutagenesis, and liquid chromatography-mass spectrometry (LC-MS) with tandem MS (MS/MS) of the tryptic peptide containing the intramolecular disulfide bond. The disulfide bond inhibits protein splicing, and splicing can be induced by reducing agents such as tris(2-carboxyethyl)phosphine (TCEP). The stability of the intramolecular disulfide bond is enhanced by electrostatic interactions between the N- and C-exteins but is reduced by elevated temperature. The presence of this intramolecular disulfide bond may contribute to the redox control of splicing activity in hypoxia and at low temperature and point to the intriguing possibility that inteins may act as switches to control extein function.

Structural and mutational studies of a hyperthermophilic intein from DNA polymerase II of Pyrococcus abyssi.

Protein splicing is a precise self-catalyzed process in which an intein excises itself from a precursor with the concomitant ligation of the flanking polypeptides (exteins). Protein splicing proceeds through a four-step reaction but the catalytic mechanism is not fully understood at the atomic level. We report the solution NMR structures of the hyperthermophilic Pyrococcus abyssi PolII intein, which has a noncanonical C-terminal glutamine instead of an asparagine. The NMR structures were determined to a backbone root mean square deviation of 0.46 Å and a heavy atom root mean square deviation of 0.93 Å. The Pab PolII intein has a common HINT (hedgehog intein) fold but contains an extra ß-hairpin that is unique in the structures of thermophilic inteins. The NMR structures also show that the Pab PolII intein has a long and disordered loop in place of an endonuclease domain. The N-terminal Cys-1 amide is hydrogen bonded to the Thr-90 hydroxyl in the conserved block-B TXXH motif and the Cys-1 thiol forms a hydrogen bond with the block F Ser-166. Mutating Thr-90 to Ala dramatically slows N-terminal cleavage, supporting its pivotal role in promoting the N-S acyl shift. Mutagenesis also showed that Thr-90 and His-93 are synergistic in catalyzing the N-S acyl shift. The block F Ser-166 plays an important role in coordinating the steps of protein splicing. NMR spin relaxation indicates that the Pab PolII intein is significantly more rigid than mesophilic inteins, which may contribute to the higher optimal temperature for protein splicing.

1H, 13C, and 15N NMR assignments of the Pyrococcus abyssi DNA polymerase II intein.

Protein splicing is a precise post-translational process mediated by inteins. Inteins are intervening proteins that cleave themselves from a precursor protein while joining the flanking sequences. Here we report the (15)N, (13)C, and (1)H chemical shift assignments of the intein from DNA polymerase II of Pyrococcus abyssi (Pab PolII intein), which has been recombinantly overexpressed and isotopically labeled. The NMR assignments of Pab PolII intein are essential for solution structure determination and protein dynamics study.

Canonical protein splicing of a class 1 intein that has a class 3 noncanonical sequence motif.

A Thermobifida fusca intein has two characteristics of class 3 inteins: a noncontiguous covariant Trp-Cys-Thr triplet and a Ser flanking its C terminus. However, it has Cys at position one, characteristic of class 1 inteins. Splicing does not require the internal Cys, which may instead coordinate the active site. Therefore, the intein is class 1.

Mechanism of protein splicing of the Pyrococcus abyssi lon protease intein.

Protein splicing is a post-translational process by which an intervening polypeptide, the intein, excises itself from the flanking polypeptides, the exteins, coupled to ligation of the exteins. The lon protease of Pyrococcus abyssi (Pab) is interrupted by an intein. When over-expressed as a fusion protein in Escherichia coli, the Pab lon protease intein can promote efficient protein splicing. Mutations that block individual steps of splicing generally do not lead to unproductive side reactions, suggesting that the intein tightly coordinates the splicing process. The intein can splice, although it has Lys in place of the highly conserved penultimate His, and mutants of the intein in the C-terminal region lead to the accumulation of stable branched-ester intermediate.

Protein splicing of the three Pyrococcus abyssi ribonucleotide reductase inteins.

An intein is a polypeptide that interrupts the functional domains of a protein, called the exteins. The intein can facilitate its own excision from the exteins, concomitant with the ligation of the exteins, in a process called protein splicing. The alpha subunit of the ribonucleotide reductase of the extreme thermophile Pyrococcus abyssi is interrupted by three inteins in separate insertion sites. Each intein can facilitate protein splicing when over-expressed in Escherichia coli, with affinity domains serving as the exteins. The influence of the N-terminal flanking residue on the efficiency of splicing is specific to each intein. Each intein has a different downstream nucleophilic residue, and cannot tolerate substitution to a residue of lesser or equal nucleophilicity. The influence of the conserved penultimate His also differs between the inteins.