Crystal, Solution and In silico Structural Studies of Dihydrodipicolinate Synthase from the Common Grapevine

Dihydrodipicolinate synthase (DHDPS) catalyzes the rate limiting step in lysine biosynthesis in bacteria and plants. The structure of DHDPS has been determined from several bacterial species and shown in most cases to form a homotetramer or dimer of dimers. However, only one plant DHDPS structure has been determined to date from the wild tobacco species, Nicotiana sylvestris (Blickling et al. (1997) J. Mol. Biol. 274, 608–621). Whilst N. sylvestris DHDPS also forms a homotetramer, the plant enzyme adopts a ‘back-to-back’ dimer of dimers compared to the ‘head-to-head’ architecture observed for bacterial DHDPS tetramers. This raises the question of whether the alternative quaternary architecture observed for N. sylvestris DHDPS is common to all plant DHDPS enzymes. Here, we describe the structure of DHDPS from the grapevine plant, Vitis vinifera, and show using analytical ultracentrifugation, small-angle X-ray scattering and X-ray crystallography that V. vinifera DHDPS forms a ‘back-to-back’ homotetramer, consistent with N. sylvestris DHDPS. This study is the first to demonstrate using both crystal and solution state measurements that DHDPS from the grapevine plant adopts an alternative tetrameric architecture to the bacterial form, which is important for optimizing protein dynamics as suggested by molecular dynamics simulations reported in this study

From knock-out phenotype to three-dimensional structure of a promising antibiotic target from streptococcus pneumoniae

Given the rise in drug-resistant Streptococcus pneumoniae, there is an urgent need to discover new antimicrobials targeting this pathogen and an equally urgent need to characterize new drug targets. A promising antibiotic target is dihydrodipicolinate synthase (DHDPS), which catalyzes the rate-limiting step in lysine biosynthesis. In this study, we firstly show by gene knock out studies that S. pneumoniae (sp) lacking the DHDPS gene is unable to grow unless supplemented with lysine-rich media. We subsequently set out to characterize the structure, function and stability of the enzyme drug target. Our studies show that sp-DHDPS is folded and active with a kcat = 22 s-1 , KM PYR = 2.55 ± 0.05 mM and KM ASA = 0.044 ± 0.003 mM. Thermal denaturation experiments demonstrate sp-DHDPS exhibits an apparent melting temperature (TM app) of 72 °C, which is significantly greater than Escherichia coli DHDPS (Ec-DHDPS) (TM app = 59 °C). Sedimentation studies show that sp-DHDPS exists in a dimer-tetramer equilibrium with a KD 4→2 = 1.7 nM, which is considerably tighter than its E. coli ortholog (KD 4→2 = 76 nM). To further characterize the structure of the enzyme and probe its enhanced stability, we solved the high resolution (1.9 Å) crystal structure of sp-DHDPS (PDB ID 3VFL). The enzyme is tetrameric in the crystal state, consistent with biophysical measurements in solution. Although the sp-DHDPS and Ec-DHDPS active sites are almost identical, the tetramerization interface of the s. pneumoniae enzyme is significantly different in composition and has greater buried surface area (800 Å2 ) compared to its E. coli counterpart (500 Å2 ). This larger interface area is consistent with our solution studies demonstrating that sp-DHDPS is considerably more thermally and thermodynamically stable than Ec-DHDPS

Identification of the evolutionary divergence in DHDPS and DHDPR

DHDPS and DHDPR are the first two committed steps in the DAP pathway: a pathway responsible for the biosynthesis of lysine. It is only present in bacteria and plants making an important biological target. While DHDPS exists in a homotetrameric “dimer of dimers” formation in both bacteria and plants, the arrangement of monomers is different. In bacteria, the dimers face toward each other in a front to front arrangement. However, in plants, the orientation of the dimers is flipped into a back to back arrangement. An evolutionary difference is also observed in DHDPR. In bacteria, the protein exists in a homotetrameric conformation whereas in plants it has been shown to exist in a dimeric conformation. The exact reason for these differences in structure remain unclear but it is thought to due to evolutionary changes between the two organism types. In this study, a lycophyte DHDPS from Selaginella moellendorffii was found to exist in a substrate mediated equilibrium between dimer and tetramer, with no ligands bound. When the substrate pyruvate is bound to the enzyme, the equilibrium shifted to the tetrameric species. However, in the presence of the allosteric inhibitor lysine, the equilibrium was found to shift to a dimeric species in solution. This equilibrium could exist as a “missing link” in the evolution of the plant type quaternary structure of the DHDPS enzyme. Another subject of investigation was the characterisation of red, green and brown algal DHDPRs. The quaternary state of these species was found to be dimeric in nature. This corresponds to the proposed evolutionary lineage in which most of these species exist after the plant type species in the lineage. The exception to this is the green alga Chlamydomonas reinhardtii DHDPR which exists in an equilibrium between tetramer and dimer. As this organism lies in the evolutionary lineage between bacterial and plant forms, it is possible that this organisms DHDPR exists as the “divergence point” between these two species. C. reinhardtii DHDPR also contains a disulfide-dependent dimer interface. In the presence of reducing agent, the enzyme exists in an exclusively dimeric state. These evolutionary lineages could be applied to other enzyme evolution systems from the DAP pathway and beyond

Structural and dynamic requirements for optimal activity of the essential bacterial enzyme dihydrodipicolinate synthase

Dihydrodipicolinate synthase (DHDPS) is an essential enzyme involved in the lysine biosynthesis pathway. DHDPS from E. coli is a homotetramer consisting of a 'dimer of dimers', with the catalytic residues found at the tight-dimer interface. Crystallographic and biophysical evidence suggest that the dimers associate to stabilise the active site configuration, and mutation of a central dimer-dimer interface residue destabilises the tetramer, thus increasing the flexibility and reducing catalytic efficiency and substrate specificity. This has led to the hypothesis that the tetramer evolved to optimise the dynamics within the tight-dimer. In order to gain insights into DHDPS flexibility and its relationship to quaternary structure and function, we performed comparative Molecular Dynamics simulation studies of native tetrameric and dimeric forms of DHDPS from E. coli and also the native dimeric form from methicillin-resistant Staphylococcus aureus (MRSA). These reveal a striking contrast between the dynamics of tetrameric and dimeric forms. Whereas the E. coli DHDPS tetramer is relatively rigid, both the E. coli and MRSA DHDPS dimers display high flexibility, resulting in monomer reorientation within the dimer and increased flexibility at the tight-dimer interface. The mutant E. coli DHDPS dimer exhibits disorder within its active site with deformation of critical catalytic residues and removal of key hydrogen bonds that render it inactive, whereas the similarly flexible MRSA DHDPS dimer maintains its catalytic geometry and is thus fully functional. Our data support the hypothesis that in both bacterial species optimal activity is achieved by fine tuning protein dynamics in different ways: E. coli DHDPS buttresses together two dimers, whereas MRSA dampens the motion using an extended tight-dimer interface

A single molecule study of the GroEL active cage mechanism

The cylindrical chaperonin GroEL and its lid-shaped cofactor GroES of Escherichia coli perform an essential role in assisting protein folding by transiently encapsulating non-native substrate in an ATP-regulated mechanism. It remains controversial whether the chaperonin system functions solely as an infinite dilution chamber, preventing off-pathway aggregation, or actively enhances folding kinetics by modulating the folding energy landscape. Here we developed single-molecule approaches to distinguish between passive and active chaperonin mechanisms. Using low protein concentrations to exclude aggregation, in combination with highly sensitive spectroscopic methods, such as single-molecule Förster resonance energy transfer (FRET) and fluorescence correlation spectroscopy (FCS), we measured the spontaneous and GroEL/ES-assisted folding of double-mutant maltose binding protein (DM-MBP), and a natural GroEL substrate - dihydrodipicolinate synthase (DapA). We show that both proteins form highly flexible, kinetically trapped folding intermediates, when folding in free solution and do not engage in inter-molecular interactions, such as aggregation, at sufficiently low concentration. We find that in the absence of aggregation, GroEL/ES accelerates folding of DM-MBP up to 8-fold over the spontaneous folding rate. The folding of DapA could be measured at physiological temperature and was found to be ~130-fold accelerated by GroEL/ES. As accelerated folding was independent of repetitive cycles of protein binding and release from GroEL, we demonstrate that iterative annealing does not significantly contribute to chaperonin assisted substrate folding. With a single molecule FRET based approach, we show that a given substrate molecule spends most of the time (~80%) during the GroEL reaction cycle inside the GroEL central cavity, in line with the inner GroEL cage being the active principle in folding catalysis. Moreover, photoinduced electron transfer experiments on DM-MBP provided direct experimental evidence that the confining environment of the chaperonin cage restricts polypeptide chain dynamics. This effect is mainly mediated by the net-negatively charged wall of the GroEL/ES cavity, as shown using the GroEL mutant EL(KKK2) in which the net-negative charge is removed. Taken together, we were able to develop novel approaches, based on single molecule spectroscopy and making use of GroEL as a single molecule sorting machine, to measure GroEL substrate folding rates at sub-nanomolar concentrations. We also, for the first time, provide direct experimental evidence of conformational restriction of an encapsulated polypeptide in a chaperonin cage. Our findings suggest that global encapsulation inside the GroEL/ES cavity, not iterative cycles of annealing and forced unfolding, can accelerate substrate folding by reduction of an entropic energy barrier to the folded state, in strong support of an active chaperonin mechanism. Accelerated folding is biologically significant as it adjusts folding rates relative to the rate of protein synthesis

Novel inhibitors of dihydrodipicolinate synthase

Dihydrodipicolinate synthase (DHDPS) catalyzes the first committed step of L-lysine and meso-diaminopimelate biosynthesis, which is the condensation of (S)-aspartate-β-semialdehyde (ASA) and pyruvate into dihydrodipicolinate via an unstable heterocyclic intermediate, (4S)-hydroxy-2,3,4,5-tetrahydro-(2S)-dipicolinic acid. DHDPS has been an attractive antibiotic target because L-lysine and meso-diaminopimelate are cross-linking components between peptidoglycan heteropolysaccharide chains in bacterial cell walls. Studies revealed that mutant auxotrophs for diaminopimelate undergo lysis in the absence of diaminopimelate in the medium; therefore the assumption is that strong inhibition of DHDPS would result in disruption of meso-diaminopimelate and L-lysine biosynthesis in bacteria and would stop or decrease bacterial growth (eventually leading to bacterial death). In this work, the DHDPS inhibitor design is focused on the allosteric site of the enzyme. It was proposed that a compound mimicking binding of two L-lysine molecules at the allosteric site at the enzyme’s dimer-dimer interface would be a more potent inhibitor than the natural allosteric inhibitor of this enzyme, L-lysine. This inhibitor (R,R-bislysine) was synthesized as a racemic mixture, which was then separated with the aid of chiral HPLC. The mechanism of feedback inhibition of DHDPS from Campylobacter jejuni with its natural allosteric modulator, L-lysine, and its synthetic mimic, R,R-bislysine, is studied in detail. It is found that L-lysine is a partial uncompetitive inhibitor with respect to pyruvate and a partial mixed inhibitor with respect to ASA. R,R-bislysine is a mixed partial inhibitor with respect to pyruvate and a noncompetitive partial inhibitor with respect to ASA, with an inhibition constant of 200 nM. Kinetic evaluation of each DHDPS mutants (Y110F, H56A, H56N, H59A and H59N) has revealed amino acids responsible for the inhibitory effect of L-lysine, R,R-bislysine, and we have found that R,R-bislysine is a strong submicromolar inhibitor of Y110F, H56A, H56N and H59N

A single molecule study of the GroEL active cage mechanism

The cylindrical chaperonin GroEL and its lid-shaped cofactor GroES of Escherichia coli perform an essential role in assisting protein folding by transiently encapsulating non-native substrate in an ATP-regulated mechanism. It remains controversial whether the chaperonin system functions solely as an infinite dilution chamber, preventing off-pathway aggregation, or actively enhances folding kinetics by modulating the folding energy landscape. Here we developed single-molecule approaches to distinguish between passive and active chaperonin mechanisms. Using low protein concentrations to exclude aggregation, in combination with highly sensitive spectroscopic methods, such as single-molecule Förster resonance energy transfer (FRET) and fluorescence correlation spectroscopy (FCS), we measured the spontaneous and GroEL/ES-assisted folding of double-mutant maltose binding protein (DM-MBP), and a natural GroEL substrate - dihydrodipicolinate synthase (DapA). We show that both proteins form highly flexible, kinetically trapped folding intermediates, when folding in free solution and do not engage in inter-molecular interactions, such as aggregation, at sufficiently low concentration. We find that in the absence of aggregation, GroEL/ES accelerates folding of DM-MBP up to 8-fold over the spontaneous folding rate. The folding of DapA could be measured at physiological temperature and was found to be ~130-fold accelerated by GroEL/ES. As accelerated folding was independent of repetitive cycles of protein binding and release from GroEL, we demonstrate that iterative annealing does not significantly contribute to chaperonin assisted substrate folding. With a single molecule FRET based approach, we show that a given substrate molecule spends most of the time (~80%) during the GroEL reaction cycle inside the GroEL central cavity, in line with the inner GroEL cage being the active principle in folding catalysis. Moreover, photoinduced electron transfer experiments on DM-MBP provided direct experimental evidence that the confining environment of the chaperonin cage restricts polypeptide chain dynamics. This effect is mainly mediated by the net-negatively charged wall of the GroEL/ES cavity, as shown using the GroEL mutant EL(KKK2) in which the net-negative charge is removed. Taken together, we were able to develop novel approaches, based on single molecule spectroscopy and making use of GroEL as a single molecule sorting machine, to measure GroEL substrate folding rates at sub-nanomolar concentrations. We also, for the first time, provide direct experimental evidence of conformational restriction of an encapsulated polypeptide in a chaperonin cage. Our findings suggest that global encapsulation inside the GroEL/ES cavity, not iterative cycles of annealing and forced unfolding, can accelerate substrate folding by reduction of an entropic energy barrier to the folded state, in strong support of an active chaperonin mechanism. Accelerated folding is biologically significant as it adjusts folding rates relative to the rate of protein synthesis