Evidence of Allosteric Enzyme Regulation via Changes in Conformational Dynamics: A Hydrogen/Deuterium Exchange Investigation of Dihydrodipicolinate Synthase.
Dihydrodipicolinate synthase is a tetrameric enzyme of the diaminopimelate pathway in bacteria and plants. The protein catalyzes the condensation of pyruvate (Pyr) and aspartate semialdehyde en route to the end product lysine (Lys). Dihydrodipicolinate synthase from Campylobacter jejuni (CjDHDPS) is allosterically inhibited by Lys. CjDHDPS is a promising antibiotic target, as highlighted by the recent development of a potent bis-lysine (bisLys) inhibitor. The mechanism whereby Lys and bisLys allosterically inhibit CjDHDPS remains poorly understood. In contrast to the case for other allosteric enzymes, crystallographically detectable conformational changes in CjDHDPS upon inhibitor binding are very minor. Also, it is difficult to envision how Pyr can access the active site; the available X-ray data seemingly imply that each turnover step requires diffusion-based mass transfer through a narrow access channel. This study employs hydrogen/deuterium exchange mass spectrometry for probing the structure and dynamics of CjDHDPS in a native solution environment. The deuteration kinetics reveal that the most dynamic protein regions are in the direct vicinity of the substrate access channel. This finding is consistent with the view that transient opening/closing fluctuations facilitate access of the substrate to the active site. Under saturating conditions, both Lys and bisLys cause dramatically reduced dynamics in the inhibitor binding region. In addition, rigidification extends to regions close to the substrate access channel. This finding strongly suggests that allosteric inhibitors interfere with conformational fluctuations that are required for CjDHDPS substrate turnover. In particular, our data imply that Lys and bisLys suppress opening/closing events of the access channel, thereby impeding diffusion of the substrate into the active site. Overall, this work illustrates why allosteric control does not have to be associated with crystallographically detectable large-scale transitions. Our experiments provide evidence that in CjDHDPS allostery is mediated by changes in the extent of thermally activated conformational fluctuations.
Department of Chemistry, The University of Western Ontario , London, Ontario N6A 5B7, Canada.
Modupeola A
MA
Sowole
Department of Chemistry, University of Saskatchewan , 110 Science Place, Saskatoon, Saskatchewan S7N 5C9, Canada.
Sarah
S
Simpson
Department of Chemistry, University of Saskatchewan , 110 Science Place, Saskatoon, Saskatchewan S7N 5C9, Canada.
Yulia V
YV
Skovpen
Department of Chemistry, University of Saskatchewan , 110 Science Place, Saskatoon, Saskatchewan S7N 5C9, Canada.
David R J
DR
Palmer
Department of Chemistry, The University of Western Ontario , London, Ontario N6A 5B7, Canada.
Lars
L
Konermann
D000494:Allosteric Regulation; D018360:Crystallography, X-Ray; D003903:Deuterium / Q000737:chemistry; D006836:Hydro-Lyases / Q000737:chemistry*; D006859:Hydrogen / Q000737:chemistry; D011487:Protein Conformation
D006859:Hydrogen; D003903:Deuterium; D006836:Hydro-Lyases; C020040:4-hydroxy-tetrahydrodipicolinate synthase