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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.

SUBMITTER: Du Z

PROVIDER: S-EPMC2737186 | biostudies-literature | 2009 Aug

REPOSITORIES: biostudies-literature

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

Du Zhenming Z Shemella Philip T PT Liu Yangzhong Y McCallum Scott A SA Pereira Brian B Nayak Saroj K SK Belfort Georges G Belfort Marlene M Wang Chunyu C

Journal of the American Chemical Society 20090801 32

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) ...[more]

PMID: 19630416

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Project description:Proline utilization A from Escherichia coli (EcPutA) is a multifunctional flavoenzyme that oxidizes proline to glutamate through proline dehydrogenase (PRODH) and ?1-pyrroline-5-carboxylate dehydrogenase (P5CDH) activities, while also switching roles as a DNA-bound transcriptional repressor and a membrane-bound catabolic enzyme. This phenomenon, termed functional switching, occurs through a redox-mediated mechanism in which flavin reduction triggers a conformational change that increases EcPutA membrane binding affinity. Structural studies have shown that reduction of the FAD cofactor causes the ribityl moiety to undergo a crankshaft motion, indicating that the orientation of the ribityl chain is a key element of PutA functional switching. Here, we test the role of a conserved histidine that bridges from the FAD pyrophosphate to the backbone amide of a conserved leucine residue in the PRODH active site. An EcPutA mutant (H487A) was characterized by steady-state and rapid-reaction kinetics, and cell-based reporter gene experiments. The catalytic activity of H487A is severely diminished (>50-fold) with membrane vesicles as the electron acceptor, and H487A exhibits impaired lipid binding and in vivo transcriptional repressor activity. Rapid-reaction kinetic experiments demonstrate that H487A is 3-fold slower than wild-type EcPutA in a conformational change step following reduction of the FAD cofactor. Furthermore, the reduction potential (Em) of H487A is ?40 mV more positive than that of wild-type EcPutA, and H487A has an attenuated ability to catalyze the reverse PRODH chemical step of reoxidation by P5C. In this process, significant red semiquinone forms in contrast to the same reaction with wild-type EcPutA, in which facile two-electron reoxidation occurs without the formation of a measurable amount of semiquinone. These results indicate that His487 is critically important for the proline/P5C chemical step, conformational change kinetics, and functional switching in EcPutA.

Project description:UnlabelledCellular immunity is pivotal in HIV-1 pathogenesis but is hampered by viral sequence diversity. An approach to minimize this diversity is to focus immunity on conserved proteome sequences; therefore, we selected four relatively conserved regions (Gag amino acids 148 to 214 and 250 to 335, Env amino acids 521 to 606, and Nef amino acids 106 to 148), each created in three mosaics, to provide better coverage of M-group HIV-1 sequences. A conserved-region vaccine (CRV) delivering genes for these four regions as equal mixtures of three mosaics each (each region at a separate injection site) was compared to a whole-protein vaccine (WPV) delivering equimolar amounts of genes for whole Gag, Env, and Nef as clade B consensus sequences (separate injection sites). Three rhesus macaques were vaccinated via three DNA primes and a recombinant adenovirus type 5 boost (weeks 0, 4, 8, and 24, respectively). Although CRV inserts were about one-fifth that of WPV, the CRV generated comparable-magnitude blood CD4+ and CD8+ T lymphocyte responses against Gag, Env, and Nef. WPV responses preferentially targeted proteome areas outside the selected conserved regions in direct proportion to sequence lengths, indicating similar immunogenicities for the conserved regions and the outside regions. The CRV yielded a conserved-region targeting density that was approximately 5-fold higher than that of the WPV. A similar pattern was seen for bronchoalveolar lymphocytes, but with quadruple the magnitudes seen in blood. Overall, these findings demonstrate that the selected conserved regions are highly immunogenic and that anatomically isolated vaccinations with these regions focus immunodominance compared to the case for full-length protein vaccination.ImportanceHIV-1 sequence diversity is a major barrier limiting the capability of cellular immunity to contain infection and the ability of vaccines to match circulating viral sequences. To date, vaccines tested in humans have delivered whole proteins or genes for whole proteins, and it is unclear whether including only conserved sequences would yield sufficient cellular immunogenicity. We tested a vaccine delivering genes for four small conserved HIV-1 regions compared to a control vaccine with genes for whole Gag, Env, and Nef. Although the conserved regions ranged from 43 to 86 amino acids and comprised less than one-fifth of the whole Gag/Env/Nef sequence, the vaccines elicited equivalent total magnitudes of both CD4+ and CD8+ T lymphocyte responses. These data demonstrate the immunogenicity of these small conserved regions and the potential for a vaccine to steer immunodominance toward conserved epitopes.

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

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

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