Highly conserved histidine plays a dual catalytic role in protein splicing: a pKa shift mechanism

Abstract

Protein splicing is a precise autocatalytic process in which an intein excises itself from a precursor with the concomitant ligation of the flanking sequences. Protein splicing occurs through acid-base catalysis in which the ionization states of active site residues are crucial to the reaction mechanism. In inteins, several conserved histidines have been shown to play important roles in protein splicing, including the most conserved "B-block" histidine. In this study, we have combined NMR pK(a) determination with quantum mechanics/molecular mechanics (QM/MM) modeling to study engineered inteins from Mycobacterium tuberculosis (Mtu) RecA intein. We demonstrate a dramatic pK(a) shift for the invariant B-block histidine, the most conserved residue among inteins. The B-block histidine has a pK(a) of 7.3 +/- 0.6 in a precursor and a pK(a) of <3.5 in a spliced intein. The pK(a) values and QM/MM data suggest that the B-block histidine has a dual role in the acid-base catalysis of protein splicing. This histidine likely acts as a general base to initiate splicing with an acyl shift and then as a general acid to cause the breakdown of the scissile bond at the N-terminal splicing junction. The proposed pK(a) shift mechanism accounts for the biochemical data supporting the essential role for the B-block histidine and for the near absolute sequence conservation of this residue.

Figure 1

Four steps of protein splicing. During the four-step reaction, the intein (shown in red) is excised from the N-extein (shown in green) and C-extein (shown in blue), while the N-extein and C-exteins are ligated to form the mature protein.

Figure 2

B-block histidine is the most conserved residue in the intein sequences. Structure-based multiple sequence alignment of ΔΔIhh-SM and other inteins was achieved the using DALI server. ΔΔIhh-SM is an engineered and minimized Mtu RecA intein (see text). The locations of the conserved blocks A, B, F, and G are indicated above the sequences and the corresponding residues are colored in yellow. The conserved histidines are colored in magenta while other key residues are colored in cyan. H73, H429, H439 are the conserved B-block, F-block and penultimate histidines, respectively. The first C-extein residue is colored in green. The gap (▼;) in the residue numbering for ΔΔIhh-SM results from the deletion of the endonuclease domain of Mtu RecA intein.

Figure 3

Crystal structure of a minimized Mtu RecA intein, ΔΔIhh-SM (pdb code 2IN0). A. The overall horseshoe fold of HINT domains. Conserved residues, C1, H73, D422, H429, H439 and N440, are shown in stick representation. B. Hydrogen bond network between conserved residues.

Figure 4

Inhibition of protein splicing by mutations of conserved histidines (B-block histidine H73A, F-block histidine H429A and penultimate histidine H439A). A. SDS-PAGE of in vivo splicing assay.B. Western blot with an anti-C-extein antibody. MW=Molecular weight marker; Ui=Uninduced; MIC=MBP-intein-C-extein fusion (71.5 kD); MC = MBP-C-extein fusion (56.1 kD); M=MBP (43.0 kD); IC-intein-C-extein fusion (28.6 kD) I =intein (15.4 kD).

Figure 5

pK_a determination of conserved histidines in spliced intein (A) and in a precursor with an N-extein, NI (B). (A) Plot of the ring ¹⁵N chemical shift versus pH for conserved histidines, B-block H73, F-block H429 in ΔΔIhh-SM. The squares and circles represent the chemical shifts of N^δ1 and N^ε2, respectively. The pK_a of H439 in the spliced intein is shown in Table I, along with pK_as of other histidines in different constructs. (B) HMQC spectrum for intein precursor NI at pH 7.1. The average chemical shift of of H73 N^δ1 and N^ε2 is 188 ppm, midway between the values expected for neutral (204.3 ppm) and charged histidines (176 ppm), suggesting that H73 has a pK_a near 7 in the precursor protein.

Figure 6

A pK_a shift mechanism for B-block histidine H73 in the N-S acyl shift, the first step of protein splicing for the Mtu RecA intein. H73 acts as a general base to deprotonate the C1 thiol, initiating the NS acyl shift. Then H73 serves as a general acid to protonate the leaving nitrogen, breaking the scissile bond and completing the N-S acyl shift. The arrows indicate the routes of electron transfer and broken lines indicate hydrogen bonds. Intermediates A-D are described in the text.

Figure 7

Reaction profile for N-S acyl shift derived from QM/MM simulations.

Figure 8

Mechanistic role of the F-block histidine and penultimate histidine.