Publications and Funding

Pelc LA, Chen Z, Gohara DW, Vogt AD, Pozzi N, Di Cera E
Biochemistry 2015 Feb;54(7):1457-64
PMID: 25664608

Abstract

Although Thr is equally represented as Ser in the human genome and as a nucleophile is as good as Ser, it is never found in the active site of the large family of trypsin-like proteases that utilize the Asp/His/Ser triad. The molecular basis of the preference of Ser over Thr in the trypsin fold was investigated with x-ray structures of the thrombin mutant S195T free and bound to an irreversible active site inhibitor. In the free form, the methyl group of T195 is oriented toward the incoming substrate in a conformation seemingly incompatible with productive binding. In the bound form, the side chain of T195 is reoriented for efficient substrate acylation without causing steric clash within the active site. Rapid kinetics prove that this change is due to selection of an active conformation from a pre-existing ensemble of reactive and unreactive rotamers whose relative distribution determines the level of activity of the protease. Consistent with these observations, the S195T substitution is associated with a weak yet finite activity that allows identification of an unanticipated important role for S195 as the end point of allosteric transduction in the trypsin fold. The S195T mutation abrogates the Na(+)-dependent enhancement of catalytic activity in thrombin, activated protein C, and factor Xa and significantly weakens the physiologically important allosteric effects of thrombomodulin on thrombin and of cofactor Va on factor Xa. The evolutionary selection of Ser over Thr in trypsin-like proteases was therefore driven by the need for high catalytic activity and efficient allosteric regulation.

Moustafa IM, Korboukh VK, Arnold JJ, Smidansky ED, Marcotte LL, Gohara DW, Yang X, Sánchez-Farrán MA, Filman D, Maranas JK, Boehr DD, Hogle JM, Colina CM, Cameron CE
J. Biol. Chem. 2014 Dec;289(52):36229-48
PMID: 25378410

Abstract

RNA viruses encoding high- or low-fidelity RNA-dependent RNA polymerases (RdRp) are attenuated. The ability to predict residues of the RdRp required for faithful incorporation of nucleotides represents an essential step in any pipeline intended to exploit perturbed fidelity as the basis for rational design of vaccine candidates. We used x-ray crystallography, molecular dynamics simulations, NMR spectroscopy and pre-steady-state kinetics to compare a mutator (H273R) RdRp from poliovirus to the wild-type (WT) enzyme. We show that the nucleotide-binding site toggles between the nucleotide binding-occluded and nucleotide binding-competent states. The conformational dynamics between these states were enhanced by binding to primed template RNA. For the WT, the occluded conformation was favored; for H273R, the competent conformation was favored. The resonance for Met-187 in our NMR spectra reported on the ability of the enzyme to check the correctness of the bound nucleotide. Kinetic experiments were consistent with the conformational dynamics contributing to the established pre-incorporation conformational change and fidelity checkpoint. For H273R, residues comprising the active site spent more time in the catalytically competent conformation and were more positively correlated than the WT. We propose that by linking the equilibrium between the binding-occluded and binding-competent conformations of the nucleotide-binding pocket and other active-site dynamics to the correctness of the bound nucleotide, faithful nucleotide incorporation is achieved. These studies underscore the need to apply multiple biophysical and biochemical approaches to the elucidation of the physical basis for polymerase fidelity.

Davis AR, Gohara DW, Yap MN
Proc. Natl. Acad. Sci. U.S.A. 2014 Oct;111(43):15379-84
PMID: 25313041

Abstract

The prevailing “plug-in-the-bottle” model suggests that macrolide antibiotics inhibit translation by binding inside the ribosome tunnel and indiscriminately arresting the elongation of every nascent polypeptide after the synthesis of six to eight amino acids. To test this model, we performed a genome-wide analysis of translation in azithromycin-treated Staphylococcus aureus. In contrast to earlier predictions, we found that the macrolide does not preferentially induce ribosome stalling near the 5′ end of mRNAs, but rather acts at specific stalling sites that are scattered throughout the entire coding region. These sites are highly enriched in prolines and charged residues and are strikingly similar to other ligand-independent ribosome stalling motifs. Interestingly, the addition of structurally related macrolides had dramatically different effects on stalling efficiency. Our data suggest that ribosome stalling can occur at a surprisingly large number of low-complexity motifs in a fashion that depends only on a few arrest-inducing residues and the presence of a small molecule inducer.

Heyduk E, Heyduk T
Biochemistry 2014 Jan;53(2):282-92
PMID: 24359527

Abstract

Promoter melting by bacterial RNA polymerase is a key step in transcription initiation. We used a next generation sequencing (NGS) based approach to analyze in parallel promoter melting of all 4096 sequence variants of the 6 bp -10 promoter element. We used NGS read count for each sequence of a promoter library containing a randomized -10 sequence as an observable to determine relative enrichment of -10 element sequence variants at different time points of the promoter melting reaction. The analysis reinforced the dominating role of consensus bases at positions -11 and -7, demonstrated an enhanced preference for A at -11 among sequences exhibiting the fastest melting kinetics, and showed higher overall importance of the T at -7 compared to the A at -11 for efficient promoter melting. Sequences lacking the consensus bases at -7 or -11 could still melt fast if they contained compensatory base patterns at other positions. We observed a significant correlation between the duplex melting energy of -10 element and the kinetics of promoter melting that became more pronounced when the dominating base-specific interactions with RNAP were diminished. These observations indicate that promoter melting kinetics is determined by a combination of base-specific effects/interactions and sequence-dependent stability of DNA duplex with the former playing a dominating role. Our data show that NGS can provide a reliable, quantitative readout for a highly parallel analysis of DNA template sequence dependence of activities of proteins that bind or operate on a DNA template.

Pozzi N, Chen Z, Gohara DW, Niu W, Heyduk T, Di Cera E
J. Biol. Chem. 2013 Aug;288(31):22734-44
PMID: 23775088

Abstract

The zymogen prothrombin is composed of fragment 1 containing a Gla domain and kringle-1, fragment 2 containing kringle-2, and a protease domain containing A and B chains. The prothrombinase complex assembled on the surface of platelets converts prothrombin to thrombin by cleaving at Arg-271 and Arg-320. The three-dimensional architecture of prothrombin and the molecular basis of its activation remain elusive. Here we report the first x-ray crystal structure of prothrombin as a Gla-domainless construct carrying an Ala replacement of the catalytic Ser-525. Prothrombin features a conformation 80 Å long, with fragment 1 positioned at a 36-degree angle relative to the main axis of fragment 2 coaxial to the protease domain. High flexibility of the linker connecting the two kringles suggests multiple arrangements for kringle-1 relative to the rest of the prothrombin molecule. Luminescence resonance energy transfer measurements detect two distinct conformations of prothrombin in solution, in a 3:2 ratio, with the distance between the two kringles either fully extended (54 ± 2 Å) or partially collapsed (≤34 Å) as seen in the crystal structure. A molecular mechanism of prothrombin activation emerges from the structure. Of the two sites of cleavage, Arg-271 is located in a disordered region connecting kringle-2 to the A chain, but Arg-320 is well defined within the activation domain and is not accessible to proteolysis in solution. Burial of Arg-320 prevents prothrombin autoactivation and directs prothrombinase to cleave at Arg-271 first. Reversal of the local electrostatic potential then redirects prothrombinase toward Arg-320, leading to thrombin generation via the prethrombin-2 intermediate.

Pozzi N, Vogt AD, Gohara DW, Di Cera E
Curr. Opin. Struct. Biol. 2012 Aug;22(4):421-31
PMID: 22664096

Abstract

For over four decades, two competing mechanisms of ligand recognition–conformational selection and induced-fit–have dominated our interpretation of protein allostery. Defining the mechanism broadens our understanding of the system and impacts our ability to design effective drugs and new therapeutics. Recent kinetics studies demonstrate that trypsin-like proteases exist in equilibrium between two forms: one fully accessible to substrate (E) and the other with the active site occluded (E*). Analysis of the structural database confirms existence of the E* and E forms and vouches for the allosteric nature of the trypsin fold. Allostery in terms of conformational selection establishes an important paradigm in the protease field and enables protein engineers to expand the repertoire of proteases as therapeutics.