The objective of this work was to understand the determinants of the C1 domain binding affinity to DAG, a membrane-localized second messenger. This question is significant because of the functional roles of C1 domains as membrane-targeting modules within their parent proteins. The intrinsic affinity of C1 domains to DAG determines the cellular concentration of the second messenger that is required for the propagation of the signaling response, and has important implications for the selectivity of that response. In the specific case of PKCs, their DAG-binding affinities determine which isoform gets preferentially activated and its localization in the cell.35
To date, only one ligand-bound structure of C1 domain has been determined.21
The structure belongs to the C1B domain from PKCδ, a high-affinity DAG-binding module in complex with a water-soluble phorbol ester. Two features of the apo-and ligand-bound C1Bδ structures are pertinent to this discussion. First, the complex is stabilized by five hydrogen bonds, which involve two oxygen-containing groups of the phorbol ester and the backbone amide and carbonyl groups of C1Bδ.21
Second, the sidechain of the residue at position 252 (123 if we use the PKCα numbering scheme) is not involved in any specific interactions with the ligand.
It is the position 123 in PKCβII, a conventional PKC isoform that proved to be essential to tuning the C1 affinity to DAG. Newton’s laboratory at UCSD found that the mutation of Tyr123, which is conserved in all three conventional isoforms (), to Trp increases the in-vitro DAG affinity 33-fold. The mutation also made the C1BβII domain more responsive to DAG in vivo by altering the protein localization pattern from cytosol to juxtanuclear region under conditions of stimulated DAG production. Following the PKCβII lead, we introduced the same mutation into the C1B domain from PKCα and determined its binding affinity to DOG, a short-chain DAG analog, using solution NMR and fluorescence spectroscopy. The NMR and fluorescence titrations were carried out in the presence of DPC/DPS micelles for the dual purpose of keeping the protein-ligand complexes soluble and providing them with a membrane-mimicking environment. The binding curves of show that the Y123W mutation increases the DOG affinity of C1Bα at least 100-fold. Our data indicate that the mutation of Tyr123 to Trp converts C1Bα from low- to high-affinity DOG-binding module and that this property is shared by PKCα and PKCβ isoforms.
Having established a functional signature of the Y123W mutation in the form of altered DOG binding affinity, we characterized its effect on the structure and dynamics of C1Bα. The results of the chemical shift perturbation analysis and 1
measurements indicate that the mutation imposes a minimum structural perturbation on C1Bα in both apo- and DOG-bound forms. In the DOG-bound forms, we found that the C1Bα regions that are affected by ligand binding encompass the entire β12 and β34 loops (). Our data on the regions affected by ligand binding are in general agreement with the results of NMR binding studies on related DAG-responsive C1 domains in detergents.22,24
The favorable DOG-binding regime of C1Bα has enabled us to follow the titration behavior of individual residues and determine the dissociation constant.
In the absence of ligand, the interaction surface of Y123W C1Bα with detergent micelles is considerably larger than that of the wt. This surface includes the entire top half of the mutant protein as compared with only loop β34 in the wt (). A plausible explanation is that Y123W C1Bα has higher affinity to ligand-free micelles than the wt because of the well-documented propensity of Trp to partition readily into the interfacial membrane regions.36,37
The pivotal role of position 123 (or equivalent) in protein-membrane interactions is corroborated by the mutagenesis studies of a related C1Bδ domain. The Trp to Gly mutant of C1Bδ was unable to interact with PDBu-containing phospholipid vesicles, while its binding affinity to PDBu in the absence of bilayers remained fairly high with a Kd
of 25 nM.38
A recent stopped-flow study of C1BβII association with lipid membranes led to the proposal of the two-step binding mechanism, in which the first step involves the formation of the low-affinity protein-lipid complex.30
The binding of C1Bα to detergent micelles is intermediate on the chemical-shift timescale, which precludes the determination of Kd
values by NMR. It has been reported that the Kd
for the C1-lipid interactions in the absence of DAG and phorbol esters is on the order of 100 mM.9
Trp at position 123 can potentially increase the protein residency time at the membrane by decreasing the off-rate and thus facilitate the two-dimensional search for DAG that occurs in the second step of the binding process. Thus, we speculate that the preferential partitioning of Trp into the headgroup region of detergent micelles (and bilayers) can in part be responsible for the increased DAG affinity of the Y123W mutant.
It has been suggested for several protein systems that the sub-ns dynamics of protein backbone may play a role in the thermodynamics of ligand binding via the entropic contribution to free energy (reviewed in39
). To determine if Y123W mutation alters the flexibility of C1Bα, we characterized the sub-ns dynamics of N-H groups. The comparison of order parameters for the wt and Y123W C1Bα shown in revealed no substantial differences between the two proteins. The loop residue with the lowest value of SNH2
is the mutation site. The order parameter of residue 123 is virtually identical for the wt and mutant C1Bα, despite having a different amino acid at this position. Our data also revealed reduced R2
values for the C-terminal region comprising residues Leu150-Cys151-Gly152. This implies that the coordination site of the second Zn2+
ion, which holds together the N- and C-termini through the coordination of His102 and Cys151 side-chains, is dynamic.
In contrast to the sub-ns dynamics, the conformational dynamics of C1Bα on the μs timescale differed significantly between the wt and mutant. In both cases however, our relaxation dispersion data are consistent with the two-site exchange process between the ground and excited states. The process is fast on the chemical-shift timescale. In the wt C1Bα, among the five residues that undergo conformational exchange with a global kex
of 15400 s−1
, the largest dispersion amplitudes were observed for the Gln128-Gly129 pair. This QG motif, which is located at the C-terminal hinge of loop β34, is part of the consensus sequence in DAG-binding C1 domains.1
Mutation of Gln128 to Gly or Trp abolishes PDBu binding in the C1Bδ domain.38
A conservative mutation of Gln128 to Glu preserved the ability of C1Bα to bind PDBu but completely abolished DOG binding (M. Stewart and T. Igumenova, unpublished data).
The results of our MD simulations support the conclusion about the pivotal role of Gln128 in controlling the geometry and dynamics of the ligand-binding site. For example, in the Y123W mutant, two primary hydrogen bonds that stabilize the closed loop conformation involve the Gln128 sidechain. In both wt and Y123W C1Bα, the sidechain of Gln128 executes frequent rotameric hops often accompanied by the formation of transient hydrogen bonds (Figure S7
and ). Atypical C1 domains that are not capable of binding DAG have a four amino acid deletion in the β34 loop, and the requirement for Gln at position 128 is relaxed. Moreover, the β34 loop was reported to be rigid in the NMR studies of an atypical C1 domain.25,26
Thus, the observed conformational flexibility of the QG motif may be an important functional feature that is shared by all DAG-responsive C1 domains.
In the Y123W mutant, nine residues that belong to the N- and C-terminal hinges of β12 and β34 loops show quantifiable dispersion with a kex of 9600 s−1. The most prominent differences between the wt and Y123W C1Bα are in the β34N and β12C regions (). For residues that show non-negligible dispersion in both proteins, we interpreted the differences between their kex and Φex values as being indicative of the change in the conformational equilibrium between their respective ground and excited states. According to our estimates, the population of the excited state in the mutant increases 1.6–1.7 fold compared to the wt protein.
To evaluate the role of conformational dynamics in the DAG ligand recognition and binding, we have to consider the nature of the excited state. Based on the position of the exchanging residues in the three-dimensional structure of C1Bα, we speculate that the process detected in NMR relaxation dispersion experiments involves the transient breaking and formation of hydrogen bonds at the hinges of the ligand-binding loops. The most likely NH···O=C donor-acceptor pairs involved in this process are Gly129-His107 in the wt, and Gly129-His107 and Leu122-Thr113 in the Y123W C1Bα ().
Related to the hydrogen bond dynamics is the question of the closed-to-open loop transition. According to the results of our MD simulations, the closed loop conformation is never repopulated once the transition to the open state occurs during the 0–2 ns equilibration period (wt) or the 2–10 ns trajectories (Y123W). The open-loop conformations appear to be more energetically favorable than the closed-loop ones and therefore are likely to represent the ground states of wt and Y123W C1Bα. The differences between the loop tip distance distributions for the two proteins shown in suggest that the closed-to-open loop transition occurs on a slower timescale in the mutant than in the wt, which is in general agreement with our k−1 estimates.
The MD simulations identify three hydrogen bonds (Thr108
, and Gln128
-Pro112) whose presence stabilizes the closed state of the ligand-binding loops. These bonds are always absent in both wt and Y123W C1Bα when the loops are in the open state. The Gly129-His107 hydrogen bond is weakly correlated with the closed state of the Y123W mutant and is rarely formed in the open state of the wt C1Bα (Table S1
). The Leu122-Thr113 hydrogen bond has a probability of 0.87 ± 0.22 and 0.90 ± 0.16 in the mutant and wt, respectively. Thus, the correlation between the status of the Gly129-His107 and Leu122-Thr113 hydrogen bonds and the presence of the fully closed form with the loop tip distance of 5 Å is rather weak.
Based on the results of our MD simulations and the structure of C1Bδ in complex with phorbol ester21
, it is likely that the ground and excited states correspond to the open and partially closed conformations of the ligand-binding loops, respectively. The partially closed form may have a higher DAG affinity than the ground state with the open-loop conformation. DAG is a smaller ligand compared to the phorbol ester that has bulky polycyclic groups, and would be accommodated more effectively in a smaller inter-loop space. The secondary hydrogen bonds that involve the conserved QG motif (Gly129-His107, Gln128
-Leu122, and Tyr109-Gln128
, Table S1
) stabilize the loop conformations observed in the MD simulations, and are also likely to stabilize the excited partially closed state.
The nominal time scale of the MD simulation is shorter than the time scale of the conformational transitions that determine the chemical exchange line-broadening; nonetheless, the results of the simulation suggests that the opening and closing of the ligand binding loops may provide the mechanism for the experimentally observed chemical exchange process. Further mutagenesis studies are required to more definitively determine the nature of these motional processes. In further support of our hypotheses about the excited state, we observed that the destabilization of the closed and partially closed conformation by introducing the Q128E mutation into C1Bα (and thus eliminating hydrogen bonds in which the Gln128 sidechain serves as a hydrogen-bond donor) preserved PDBu binding but completely abolished DOG binding (M. Stewart and T. Igumenova, unpublished data).
The main differences between the excited states in the Y123W and wt C1Bα are in the β12C and β34N regions. Thr113 (β12C) and Leu122 (β34N) are involved in hydrogen-bonding interactions between the β2 and β3 strands (), and show significant dispersion in the mutant but none in the wt. According to the crystal structure of the C1Bδ-phorbol ester complex21
, the C20 O-H group of the phorbol ester, which mimics the O-H group of DAG, serves as an acceptor for the N-H of Thr113 and a donor to C=O of both Leu122 and Thr113. Transient breaking of the Leu122-Thr113 hydrogen bond can facilitate the entry of DAG into the binding site through the partial “unzipping” of the β2/β3 strands. Through this mechanism, the excited state of the Y123W mutant can potentially have higher DAG binding affinity than the excited state of the wt, where the exchange process affecting Thr113 and Leu122 is absent. The DAG binding event would then shift the conformational equilibrium between the ground and excited states towards the high-affinity excited state. This mechanism of ligand binding has been referred to as “pre-equilibrium” or selected-fit, with the implication being that the ligand selects a high-affinity conformer from an ensemble of two or more pre-existing conformers with comparable energy.40
Conformational plasticity of ligand-binding sites has been observed in other lipid-binding proteins. For example, it has been suggested that the conformational dynamics of sterol-binding protein 2 facilitates the access of ligands to the binding site and may be required for accommodating structurally diverse ligands.41
Conformational dynamics of intestinal fatty acid-binding protein have led to the “dynamic portal” hypothesis for ligand entry42,43
that has been tested recently using relaxation-dispersion experiments.44
Along with the structural features, conformational plasticity of lipid-binding proteins may be required to ensure their ligand promiscuity. For C1 domains, their ability to bind ligands other than DAG has been used to great advantage for designing inhibitors and activators.4
To conclude, structural considerations alone cannot fully explain a 100-fold change in the C1Bα affinity to DAG brought about by the Y123W mutation. Our data indicate that there are three factors that may lead to the enhancement of DAG-binding affinity in Y123W C1Bα: (1) the increased affinity of the mutant to ligand-free membranes; (2) the existence of the excited state that is capable of more efficiently capturing DAG; and (3) the increase in the population of this excited state compared to that of the wt protein. Understanding the origins of ligand binding affinity and specificity in C1 domains is important for developing ways to modulate the activity of PKCs for therapeutic and research applications. Full understanding of the molecular details of C1-DAG interactions will have to await the determination of the C1 structure in complex with DAG in the presence of a membrane mimic.