title: 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.
authors.affiliation: Biology Department, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York 12180, USA.
authors.forename: Zhenming
authors.initials: Z
authors.lastname: Du
authors.forename: Philip T
authors.initials: PT
authors.lastname: Shemella
authors.forename: Yangzhong
authors.initials: Y
authors.lastname: Liu
authors.forename: Scott A
authors.initials: SA
authors.lastname: McCallum
authors.forename: Brian
authors.initials: B
authors.lastname: Pereira
authors.forename: Saroj K
authors.initials: SK
authors.lastname: Nayak
authors.forename: Georges
authors.initials: G
authors.lastname: Belfort
authors.forename: Marlene
authors.initials: M
authors.lastname: Belfort
authors.forename: Chunyu
authors.initials: C
authors.lastname: Wang
mesh_terms: D000595:Amino Acid Sequence; D018360:Crystallography, X-Ray; D006639:Histidine / Q000737:chemistry / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D047668:Inteins; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000737:chemistry*; D019906:Nuclear Magnetic Resonance, Biomolecular; D019154:Protein Splicing; D016415:Sequence Alignment
chemical_list: D006639:Histidine