Opening of a cryptic pocket in β-lactamase increases penicillinase activity

Understanding the functional role of protein-excited states has important implications in protein design and drug discovery. However, because these states are difficult to find and study, it is still unclear if excited states simply result from thermal fluctuations and generally detract from function or if these states can actually enhance protein function. To investigate this question, we consider excited states in β-lactamases and particularly a subset of states containing a cryptic pocket which forms under the Ω-loop. Given the known importance of the Ω-loop and the presence of this pocket in at least two homologs, we hypothesized that these excited states enhance enzyme activity. Using thiol-labeling assays to probe Ω-loop pocket dynamics and kinetic assays to probe activity, we find that while this pocket is not completely conserved across β-lactamase homologs, those with the Ω-loop pocket have a higher activity against the substrate benzylpenicillin. We also find that this is true for TEM β-lactamase variants with greater open Ω-loop pocket populations. We further investigate the open population using a combination of NMR chemical exchange saturation transfer experiments and molecular dynamics simulations. To test our understanding of the Ω-loop pocket’s functional role, we designed mutations to enhance/suppress pocket opening and observed that benzylpenicillin activity is proportional to the probability of pocket opening in our designed variants. The work described here suggests that excited states containing cryptic pockets can be advantageous for function and may be favored by natural selection, increasing the potential utility of such cryptic pockets as drug targets.

While it is well established that proteins are dynamic molecules (1), it is often unclear what these dynamics mean for function. An experimentally derived structural snapshot of a protein, such as a crystal structure, is frequently assumed to represent the (highest probability, lowest energy) ground state. This snapshot is also frequently assumed to be the functional state of the protein. In fact, rigidifying the active site or increasing the probability of the ground-state conformation is often used as a design strategy for improving catalytic activity (2, 3). In opposition to this common assumption, there are several compelling examples of functionally relevant excited states (4–9). However, it is still unclear if excited states in general play a role in function.Here, we consider an important class of excited states that contain a “cryptic” pocket, or a pocket which is absent in the ligand-free, experimentally determined structure(s). These states are of particular interest because of the potential utility of cryptic pockets as drug targets (10). These pockets provide a means to drug otherwise “undruggable” proteins and a means to enhance a desired protein activity rather than just inhibit an undesired one (11, 12). One concern, however, with the use of cryptic pockets as drug targets is that it is uncertain if there is a selective pressure to maintain the existence of a given pocket or if drug binding to that pocket could be trivially evolved away. This is at least partially because it is unknown if excited states containing cryptic pockets are simply a byproduct of the dynamic nature of proteins or if they play a bigger role in protein function.Despite the many examples of systems which are known to contain cryptic pockets (13–15), their functional relevance remains unclear because these pockets are notoriously difficult to find and study. Identification of a cryptic pocket often requires simultaneous discovery of a ligand that binds to it (16). Fortunately, recent advances in computational and experimental tools allow us to better identify and study these pockets (1, 17). To increase sampling during molecular dynamics simulations, adaptive sampling methods like fluctuation amplification of specific traits (FAST) (18) and replica exchange methods like sampling water interfaces through scaled Hamiltonians (SWISH) (19) have been developed. To analyze these datasets, methods such as Markov state models (MSMs) (20) and exposons (21) have been developed. These computational tools can then be used to inform experimental methods like room temperature crystallography (22), NMR relaxation techniques (23–25), and thiol-labeling assays (26). Previous work using these methods has shown that many different kinds of proteins have cryptic pockets and that these pockets can be targeted with drugs to allosterically affect functional sites (11, 12, 27, 28). However, it is still unclear if cryptic pockets have implications for function in the absence of ligand binding.To explore the functional relevance of excited states containing cryptic pockets, we consider a set of class A β-lactamases. β-lactamases are enzymes that confer bacteria with antibiotic resistance by hydrolyzing β-lactam antibiotics such as benzylpenicillin and cefotaxime. TEM β-lactamase in particular is an established model system for studying cryptic pockets. TEM has two known and well-characterized cryptic pockets. The first, which was found serendipitously during a drug-screening campaign, is between helices 11 and 12 (16). The second, which was more recently identified in our laboratory (21), forms when the Ω-loop undocks from the protein, so we call this pocket the Ω-loop pocket (Fig. 1). The Ω-loop pocket was discovered in molecular dynamics simulations, confirmed using thiol-labeling experiments, and subsequently shown to exert control over catalysis at the adjacent active site (21). We know the Ω-loop structure is important as it is necessary for the deacylation of β-lactam antibiotics (29), and it has been previously shown that changes in Ω-loop conformations are connected to cefotaxime activity (30, 31).Open in a separate windowFig. 1.The Ω-loop pocket seen in TEM may open in other β-lactamase homologs. The structures of four β-lactamase homologs (Left) overlay well. TEM (Protein Data Base PDB]: 1xpb) is shown in green, CTX-M-9 (PDB: 1ylj) is shown in cyan, MTB (PDB: 2gdn) is shown in orange, and GNCA (PDB: 4b88) is shown in magenta. The open Ω-loop pocket structure in TEM (Right) was identified in molecular dynamics simulations.As we have also found that the Ω-loop pocket is present in CTX-M-9 β-lactamase (21), we hypothesize that this pocket may play a role in the enzyme’s function. To test this hypothesis, we first examine if the Ω-loop pocket is conserved across β-lactamase homologs and if the presence of the pocket is correlated with increased activity against classic β-lactam substrates. Here, we mean conservation of the phenomenon of cryptic pocket opening rather than conservation of the specific amino acid identities in that region of the protein. We then use activity data for TEM variants and combine NMR with molecular dynamics to gain insight into how the open Ω-loop pocket affects the hydrolysis reaction for different substrates. Finally, we design mutations to modulate the population of the open Ω-loop pocket to explicitly test whether pocket dynamics are predictive of enzymatic activity.