Low-barrier hydrogen bonds in enzyme cooperativity

Abstract

The underlying molecular mechanisms of cooperativity and allosteric regulation are well understood for many proteins with hemoglobin and aspartate transcarbamoylase serving as prototypical examples 1,2 . Binding of effectors typically causes a structural transition of the protein that is propagated through signaling pathways to remote sites involving marked changes on the tertiary and sometimes even quaternary level [1][2][3][4][5] . However, the origin of these signals and the molecular mechanism of long-range signaling at an atomic level are a field of great dispute [5][6][7][8] . The different spatial and time scales involved in signaling pathways make the experimental observation challenging, in particular the positions and movement of mobile protons are invisible to current structural methods. Here, we report the first experimental observation of fluctuating low-barrier hydrogen bonds (LBHBs) as switching elements in cooperativity pathways of multimeric enzymes. We have observed these LBHBs in ultra-high resolution ( 1 Å) X-ray crystallographic structures of two multimeric enzymes, with their assignment validated through computational calculations. Catalytic events at the active sites switch between LBHBs and ordinary hydrogen bonds in a circuit consisting of acidic side chains and water molecules, transmitting a signal through the collective repositioning of protons much in the way of an atomistic Newtons cradle. The resulting communication synchronizes catalysis in the oligomer. Our studies provide multiple lines of evidence and a working model for not only the existence of LBHBs in proteins, but also for the first connection to enzyme cooperativity. This finding opens the door for whole new principles of drug and enzyme design, where one purposefully includes sequences of residues, which enable long-range communication and thus regulation of engineered biomolecules.The structural transitions associated with signaling in proteins are mediated by dedicated cooperativity and allosteric pathways, and commonly include the rupture and formation of salt bridges and hydrogen bonds [5][6][7][8] . In principle, signaling does not require large-scale motions and can operate via proton transfer through hydrogen bonds in conjugated proton wires as observed in e.g. water 9 . The latter involves the transient formation of LBHBs, whereby a proton is shared between two heteroatoms. LBHBs arise when the pKa values of two neighboring moieties are closely matched and the interatomic distance between the two heteroatoms are smaller than the sum of their van-der-Waals radii ( 2.55 Å for O-O pairs, 2.65 Å for O-N pairs) 10 . In enzyme function, LBHBs have been proposed to be part of charge relay networks at the active site, and to lower the activation barrier for substrate turnover 11,12 . However, ever since their initial proposal, the contribution of LBHBs in enzyme catalysis or even their mere existence has been fiercely debated. While some proponents have

Structural basis for antibiotic action of the B1 antivitamin 2′-methoxy-thiamine

“…This catalytic glutamate is forced into a short, persistent LBHB with a neighboring glutamate of the signaling proton wire, both in the resting state and in complex with the substrate. This observation demonstrates that strong LBHBs in reactant states of enzyme catalysis can indeed be anticatalytic as proposed earlier 32,33 because they may freeze out overly stable, non-productive states by 'out-tuning' otherwise intimately coupled proton-transfer potentials 14 . Our recent studies in that field indicated that LBHBs are critical elements of cooperativity pathways in enzyme multimers but are not particularly stable to ensure rapid signaling in both directions of the wire 14 .…”

“…1a). The wire is thought to synchronize catalysis in the dimer by reversibly shuttling a proton involving transient low-barrier hydrogen bonds (LBHBs) 14,15 . These LBHBs are formed between a canonical glutamate (Glu411 in EcTK) that chemically activates ThDP through protonation at the N1′ atom and a neighboring, distal glutamate of the proton wire (Glu160; Fig.…”

“…Reciprocal cofactor activation in the EcTK dimer is thought to proceed by Glu411-catalyzed protonation of ThDP N1′, tautomerization of the ThDP aminopyrimidine to the corresponding imino tautomer and acid-base catalysis of the latter (intramolecularly or via a water molecule) to form the reactive thiazolium carbene/carbanion, which in turn attacks the substrate (Extended Data Fig. 2b) [14][15][16][17][18] .…”

The Two-Species Model of transketolase explains donor substrate-binding, inhibition and heat-activation

“…However, the true Hill-coefficient of TK high is likely to be higher than the reported apparent value, whereas the true Hill-coefficient of TK low is presumably lower. The most abundant dimer, TK low -TK low , may also have previously masked the recently-elucidated asymmetric structure of the TK apo-dimer 13 , given the reduced cooperativity of TPP-binding between active sites in this dimer.…”

“…It was suggested by the authors that many thiamine-dependent enzymes may function in a similar way. Recently, the expected structural asymmetry between the two monomers of apo-dimeric E. coli TK was finally observed at the proton scale and was implicated as a key feature of the proton wire and hence cooperativity between active sites 13 .…”