Examination of the nucleotide‐linked assembly mechanism of E. coli ClpA

Escherichia coli ClpA is a AAA+ (ATPase Associated with diverse cellular Activities) chaperone that catalyzes the ATP‐dependent unfolding and translocation of substrate proteins targeted for degradation by a protease, ClpP. ClpA hexamers associate with one or both ends of ClpP tetradecamers to form ClpAP complexes. Each ClpA protomer contains two nucleotide‐binding sites, NBD1 and NBD2, and self‐assembly into hexamers is thermodynamically linked to nucleotide binding. Despite a number of studies aimed at characterizing ClpA and ClpAP‐catalyzed substrate unfolding and degradation, respectively, to date the field is unable to quantify the concentration of ClpA hexamers available to interact with ClpP for any given nucleotide and total ClpA concentration. In this work, sedimentation velocity studies are used to quantitatively examine the self‐assembly of a ClpA Walker B variant in the presence of ATP. In addition to the hexamerization, we observe the formation of a previously unreported ClpA dodecamer in the presence of ATP. Further, we report apparent equilibrium constants for the formation of each ClpA oligomer obtained from direct boundary modeling of the sedimentation velocity data. The energetics of nucleotide binding to NBD1 and NBD2 are revealed by examining the dependence of the apparent association equilibrium constants on free nucleotide concentration.     

ClpA and ClpX ATPases bind simultaneously to opposite ends of ClpP peptidase to form active hybrid complexes

The Escherichia coli ATP-dependent ClpAP and ClpXP proteases are composed of a single proteolytic component, ClpP, complexed with either of the two related chaperones, ClpA or ClpX. ClpXP and ClpAP complexes interact with different specific substrates and catalyze ATP-dependent protein unfolding and degradation. In vitro in the presence of ATP or ATPgammaS, ClpA and ClpX form homomeric rings of six subunits, which bind to one or both ends of the double heptameric rings of ClpP. We have observed that, when equimolar amounts of ClpA and ClpX hexamers are added to ClpP in vitro in the presence of ATP or ATPgammaS, hybrid complexes in which ClpX and ClpA are bound to opposite ends of the same ClpP are readily formed. The distribution of homomeric and heteromeric complexes was consistent with random binding of ClpA and ClpX to the ends of ClpP. Direct demonstration of the functionality of the heteromeric complexes was obtained by electron microscopy, which allowed us to visualize substrate translocation into proteolytically inactive ClpP chambers. Starting with hybrid complexes to which protein substrates specific to ClpX or ClpA were bound, translocation of both types of substrates was shown to occur without significant redistribution of ClpA or ClpX. The stoichiometric ratios of the ClpA, ClpX, and ClpP oligomeric complexes in vivo are consistent with the predominance of heteromeric complexes in growing cells. Thus, ClpXAP is a bifunctional protease whose two ends can independently target different classes of substrates.     

Concurrent chaperone and protease activities of ClpAP and the requirement for the N-terminal ClpA ATP binding site for chaperone activity.

ClpA, a member of the Clp/Hsp100 family of ATPases, is both an ATP-dependent molecular chaperone and the regulatory component of ClpAP protease. We demonstrate that chaperone and protease activities occur concurrently in ClpAP complexes during a single round of RepA binding to ClpAP and ATP-dependent release. This result was substantiated with a ClpA mutant, ClpA(K220V), carrying an amino acid substitution in the N-terminal ATP binding site. ClpA(K220V) is unable to activate RepA, but the presence of ClpP or chemically inactivated ClpP restores its ability to activate RepA. The presence of ClpP simultaneously facilitates degradation of RepA. ClpP must remain bound to ClpA(K220V) for these effects, indicating that both chaperone and proteolytic activities of the mutant complex occur concurrently. ClpA(K220V) itself is able to form stable complexes with RepA in the presence of a poorly hydrolyzed ATP analog, adenosine 5'-O-(thiotriphosphate), and to release RepA upon exchange of adenosine 5'-O-(thiotriphosphate) with ATP. However, the released RepA is inactive in DNA binding, indicating that the N-terminal ATP binding site is essential for the chaperone activity of ClpA. Taken together, these results suggest that substrates bound to the complex of the proteolytic and ATPase components can be partitioned between release/reactivation and translocation/degradation.     

Assembly pathway of an AAA+ protein: tracking ClpA and ClpAP complex formation in real time

The ClpAP chaperone-protease complex is active as a cylindrically shaped oligomeric complex built of the proteolytic ClpP double ring as the core of the complex and two ClpA hexamers associating with the ends of the core cylinder. The ClpA chaperone belongs to the larger family of AAA+ ATPases and is responsible for preparing protein substrates for degradation by ClpP. Here, we study in real time using fluorescence and light scattering stopped-flow methods the complete assembly pathway of this bacterial chaperone-protease complex consisting of ATP-induced ClpA hexamer formation and the subsequent association of ClpA hexamers with the ClpP core cylinder. We provide evidence that ClpA assembles into hexamers via a tetrameric intermediate and that hexamerization coincides with the appearance of ATPase activity. While ATP-induced oligomerization of ClpA is a prerequisite for binding of ClpA to ClpP, the kinetics of ClpA hexamer formation are not influenced by the presence of ClpP. Models for ClpA hexamerization and ClpA-ClpP association are presented along with rate parameters obtained from numerical fitting procedures. The hexamerization kinetics show that the tetrameric intermediate transiently accumulates, forming rapidly at early time points and then decaying at a slower rate to generate the hexamer. The association of assembled ClpA hexamers with the ClpP core cylinder displays cooperativity, supporting the coexistence of interchanging ClpP conformations with different affinities for ClpA.     

Binding and degradation of heterodimeric substrates by ClpAP and ClpXP

ClpA and ClpX function both as molecular chaperones and as the regulatory components of ClpAP and ClpXP proteases, respectively. ClpA and ClpX bind substrate proteins through specific recognition signals, catalyze ATP-dependent protein unfolding of the substrate, and when in complexes with ClpP translocate the unfolded polypeptide into the cavity of the ClpP peptidase for degradation. To examine the mechanism of interaction of ClpAP with dimeric substrates, single round binding and degradation experiments were performed, revealing that ClpAP degraded both subunits of a RepA homodimer in one cycle of binding. Furthermore, ClpAP was able to degrade both protomers of a RepA heterodimer in which only one subunit contained the ClpA recognition signal. In contrast, ClpXP degraded both subunits of a dimeric substrate only when both protomers contained a recognition signal. These data suggest that ClpAP and ClpXP may recognize and bind substrates in significantly different ways.     

The ClpP N-terminus coordinates substrate access with protease active site reactivity

Energy-dependent protein degradation machines, such as the Escherichia coli protease ClpAP, require regulated interactions between the ATPase component (ClpA) and the protease component (ClpP) for function. Recent studies indicate that the ClpP N-terminus is essential in these interactions, yet the dynamics of this region remain unclear. Here, we use synchrotron hydroxyl radical footprinting and kinetic studies to characterize functionally important conformational changes of the ClpP N-terminus. Footprinting experiments show that the ClpP N-terminus becomes more solvent-exposed upon interaction with ClpA. In the absence of ClpA, deletion of the ClpP N-terminus increases the initial degradation rate of large peptide substrates 5-15-fold. Unlike ClpAP, ClpPDeltaN exhibits a distinct slow phase of product formation that is eliminated by the addition of hydroxylamine, suggesting that truncation of the N-terminus leads to stabilization of the acyl-enzyme intermediate. These results indicate that (1) the ClpP N-terminus acts as a "gate" controlling substrate access to the active sites, (2) binding of ClpA opens this "gate", allowing substrate entry and formation of the acyl-enzyme intermediate, and (3) closing of the N-terminal "gate" stimulates acyl-enzyme hydrolysis.     

The structural basis for the activation and peptide recognition of bacterial ClpP

ClpP and its ATPase compartment, ClpX or ClpA, remove misfolded proteins in cells and are of utmost importance in protein quality control. The ring hexamers of ClpA or ClpX recognize, unfold, and translocate target substrates into the degradation chamber of the double-ring tetradecamer of ClpP. The overall reaction scheme catalyzed by ClpXP or ClpAP has been proposed; however, the molecular mechanisms associated with substrate recognition and degradation have not yet been clarified in detail. To investigate these mechanisms, we determined the crystal structures of ClpP from Helicobacter pylori in complex with product peptides bound to the active site as well as in the apo state. In the complex structure, the peptides are zipped with two antiparallel strands of ClpP and point to the adjacent active site, thus providing structural explanations for the broad substrate specificity, the product inhibition and the processive degradation of substrates in the chamber. The structures also suggest that substrate binding causes local conformational changes around the active site that ultimately induce the active conformation of ClpP.     

A Single ClpS Monomer Is Sufficient to Direct the Activity of the ClpA Hexamer

ClpS is an adaptor protein that interacts with ClpA and promotes degradation of proteins with N-end rule degradation motifs (N-degrons) by ClpAP while blocking degradation of substrates with other motifs. Although monomeric ClpS forms a 1:1 complex with an isolated N-domain of ClpA, only one molecule of ClpS binds with high affinity to ClpA hexamers (ClpA₆). One or two additional molecules per hexamer bind with lower affinity. Tightly bound ClpS dissociates slowly from ClpA₆ with a t_½ of ∼3 min at 37 °C. Maximum activation of degradation of the N-end rule substrate, LR-GFP_Venus, occurs with a single ClpS bound per ClpA₆; one ClpS is also sufficient to inhibit degradation of proteins without N-degrons. ClpS competitively inhibits degradation of unfolded substrates that interact with ClpA N-domains and is a non-competitive inhibitor with substrates that depend on internal binding sites in ClpA. ClpS inhibition of substrate binding is dependent on the order of addition. When added first, ClpS blocks binding of both high and low affinity substrates; however, when substrates first form committed complexes with ClpA₆, ClpS cannot displace them or block their degradation by ClpP. We propose that the first molecule of ClpS binds to the N-domain and to an additional functional binding site, sterically blocking binding of non-N-end rule substrates as well as additional ClpS molecules to ClpA₆. Limiting ClpS-mediated substrate delivery to one per ClpA₆ avoids congestion at the axial channel and allows facile transfer of proteins to the unfolding and translocation apparatus.     

An intrinsic degradation tag on the ClpA C-terminus regulates the balance of ClpAP complexes with different substrate specificity

ATP-dependent protein degradation in bacteria is carried out by barrel-shaped proteases architecturally related to the proteasome. In Escherichia coli, ClpP interacts with two alternative ATPases, ClpA or ClpX, to form active protease complexes. ClpAP and ClpXP show different but overlapping substrate specificities. ClpXP is considered the primary recipient of ssrA-tagged substrates while ClpAP in complex with ClpS processes N-end rule substrates. Notably, in its free form, but not in complex with ClpS, ClpAP also degrades ssrA-tagged substrates and its own chaperone component, ClpA. To reveal the mechanism of ClpAP-mediated ClpA degradation, termed autodegradation, and its possible role in regulating ClpAP levels, we dissected ClpA to show that the flexible C-terminus of the second AAA module serves as the degradation signal. We demonstrate that ClpA becomes largely resistant to autodegradation in the absence of its C-terminus and, conversely, transfer of the last 11 residues of ClpA to the C-terminus of green fluorescent protein (GFP) renders GFP a substrate of ClpAP. This autodegradation tag bears similarity to the ssrA-tag in its degradation behavior, displaying similar catalytic turnover rates when coupled to GFP but a twofold lower apparent affinity constant compared to ssrA-tagged GFP. We show that, in analogy to the prevention of ssrA-mediated recognition, the adaptor ClpS inhibits autodegradation by a specificity switch as opposed to direct masking of the degradation signal. Our results demonstrate that in the presence of ssrA-tagged substrates, ClpA autodegradation will be competitively reduced. This simple mechanism allows for dynamic reallocation of free ClpAP versus ClpAPS in response to the presence of ssrA-tagged substrates.     

ClpP: a structurally dynamic protease regulated by AAA+ proteins

Proteolysis is an important process for many aspects of bacterial physiology. Clp proteases carry out a large proportion of protein degradation in bacteria. These enzymes assemble in complexes that combine the protease ClpP and the unfoldase, ClpA or ClpX. ClpP oligomerizes as two stacked heptameric rings enclosing a central chamber containing the proteolytic sites. ClpX and ClpA assemble into hexameric rings that bind both axial surfaces of the ClpP tetradecamer forming a barrel-like complex. ClpP requires association with ClpA or ClpX to unfold and thread protein substrates through the axial pore into the inner chamber where degradation occurs. A gating mechanism regulated by the ATPase exists at the entry of the ClpP axial pore and involves the N-terminal regions of the ClpP protomers. These gating motifs are located at the axial regions of the tetradecamer but in most crystal structures they are not visible. We also lack structural information about the ClpAP or ClpXP complexes. Therefore, the structural details of how the axial gate in ClpP is regulated by the ATPases are unknown. Here, we review our current understanding of the conformational changes that ClpA or ClpX induce in ClpP to open the axial gate and increase substrate accessibility into the degradation chamber. Most of this knowledge comes from the recent crystal structures of ClpP in complex with acyldepsipeptides (ADEP) antibiotics. These small molecules are providing new insights into the gating mechanism of this protease because they imitate the interaction of ClpA/ClpX with ClpP and activate its protease activity.     

Control of Substrate Gating and Translocation into ClpP by Channel Residues and ClpX Binding

ClpP is a self-compartmentalized protease, which has very limited degradation activity unless it associates with ClpX to form ClpXP or with ClpA to form ClpAP. Here, we show that ClpX binding stimulates ClpP cleavage of peptides larger than a few amino acids and enhances ClpP active-site modification. Stimulation requires ATP binding but not hydrolysis by ClpX. The magnitude of this enhancement correlates with increasing molecular weight of the molecule entering ClpP. Amino-acid substitutions in the channel loop or helix A of ClpP enhance entry of larger substrates into the free enzyme, eliminate ClpX binding in some cases, and are not further stimulated by ClpX binding in other instances. These results support a model in which the channel residues of free ClpP exclude efficient entry of all but the smallest peptides into the degradation chamber, with ClpX binding serving to relieve these inhibitory interactions. Specific ClpP channel variants also prevent ClpXP translocation of certain amino-acid sequences, suggesting that the wild-type channel plays an important role in facilitating broad translocation specificity. In combination with previous studies, our results indicate that collaboration between ClpP and its partner ATPases opens a gate that functions to exclude larger substrates from isolated ClpP.     

ClpP hydrolyzes a protein substrate processively in the absence of the ClpA ATPase: mechanistic studies of ATP-independent proteolysis

ATP-dependent proteases are processive, meaning that they degrade full-length proteins into small peptide products without releasing large intermediates along the reaction pathway. In the case of the bacterial ATP-dependent protease ClpAP, ATP hydrolysis by the ClpA component has been proposed to be required for processive proteolysis of full-length protein substrates. We present here data showing that in the absence of the ATPase subunit ClpA, the protease subunit ClpP can degrade full-length protein substrates processively, albeit at a greatly reduced rate. Moreover, the size distribution of peptide products from a ClpP-catalyzed digest is remarkably similar to the size distribution of products from a ClpAP-catalyzed digest. The ClpAP- and ClpP-generated peptide product size distributions are fitted well by a sum of multiple underlying Gaussian peaks with means at integral multiples of approximately 900 Da (7-8 amino acids). Our results are consistent with a mechanism in which ClpP controls product sizes by alternating between translocation in steps of 7-8 (+/-2-3) amino acid residues and proteolysis. On the structural and molecular level, the step size may be controlled by the spacing between the ClpP active sites, and processivity may be achieved by coupling peptide bond hydrolysis to the binding and release of substrate and products in the protease chamber.     

The N-terminal substrate-binding domain of ClpA unfoldase is highly mobile and extends axially from the distal surface of ClpAP protease

ClpAP is a barrel-like complex consisting of hexameric rings of the ClpA ATPase stacked on the double heptameric ring of ClpP peptidase. ClpA has two AAA+ domains (Dl and D2) and a 153-residue N-domain. Substrate proteins bind to the distal surface of ClpA and are unfolded and translocated axially into ClpP. To gain insight into the functional architecture of ClpA in the ATPgammaS state, we have determined its structure at 12A resolution by cryo-electron microscopy. The resulting model has two tiers, corresponding to rings of Dl and D2 domains: oddly, there is no sign of the N-domains in the density map. However, they were detected as faint diffuse density distal to the Dl tier in a difference image between wild-type ClpAP and a mutant lacking the N-domain. This region is also accentuated in a variance map of ClpAP and in a difference imaging experiment with ClpAP complexed with ClpS, a 12kDa protein that binds to the N-domain. These observations demonstrate that the N-domains are highly mobile. From molecular modeling, we identify their median position and estimate that they undergo fluctuations of at least 30A. We discuss the implications of these observations for the role of N-domains in substrate binding: either they effect an initial transient binding, relaying substrate to a second site on the Dl tier where unfolding ensues, or they may serve as an entropic brush to clear the latter site of non-specifically bound ligands or substrates bound in non-productive complexes.     

The ClpP double ring tetradecameric protease exhibits plastic ring-ring interactions, and the N termini of its subunits form flexible loops that are essential for ClpXP and ClpAP complex formation

ClpP is a conserved serine-protease with two heptameric rings that enclose a large chamber containing the protease active sites. Each ClpP subunit can be divided into a handle region, which mediates ring-ring interactions, and a head domain. ClpP associates with the hexameric ATPases ClpX and ClpA, which can unfold and translocate substrate proteins through the ClpP axial pores into the protease lumen for degradation. We have determined the x-ray structure of Streptococcus pneumoniae ClpP(A153P) at 2.5 A resolution. The structure revealed two novel features of ClpP which are essential for ClpXP and ClpAP functional activities. First, the Ala --> Pro mutation disrupts the handle region, resulting in an altered ring-ring dimerization interface, which, in conjunction with biochemical data, demonstrates the unusual plasticity of this region. Second, the structure shows the existence of a flexible N-terminal loop in each ClpP subunit. The loops line the axial pores in the ClpP tetradecamer and then protrude from the protease apical surface. The sequence of the N-terminal loop is highly conserved in ClpP across all kingdoms of life. These loops are essential determinants for complex formation between ClpP and ClpX/ClpA. Mutation of several amino acid residues in this loop or the truncation of the loop impairs ClpXP and ClpAP complex formation and prevents the coupling between ClpX/ClpA and ClpP activities.     

Both ATPase Domains of ClpA Are Critical for Processing of Stable Protein Structures

ClpA is a ring-shaped hexameric chaperone that binds to both ends of the protease ClpP and catalyzes the ATP-dependent unfolding and translocation of substrate proteins through its central pore into the ClpP cylinder. Here we study the relevance of ATP hydrolysis in the two ATPase domains of ClpA. We designed ClpA Walker B variants lacking ATPase activity in the first (D1) or the second ATPase domain (D2) without impairing ATP binding. We found that the two ATPase domains of ClpA operate independently even in the presence of the protease ClpP or the adaptor protein ClpS. Notably, ATP hydrolysis in the first ATPase module is sufficient to process a small, single domain protein of low stability. Substrate proteins of moderate local stability were efficiently processed when D1 was inactivated. However, ATP hydrolysis in both domains was required for efficiently processing substrates of high local stability. Furthermore, we provide evidence for the ClpS-dependent directional translocation of N-end rule substrates from the N to C terminus and propose a mechanistic model for substrate handover from the adaptor protein to the chaperone.     

The molecular chaperone, ClpA, has a single high affinity peptide binding site per hexamer

Substrate recognition by Clp chaperones is dependent on interactions with motifs composed of specific peptide sequences. We studied the binding of short motif-bearing peptides to ClpA, the chaperone component of the ATP-dependent ClpAP protease of Escherichia coli in the presence of ATPgammaS and Mg2+ at pH 7.5. Binding was measured by isothermal titration calorimetry (ITC) using the peptide, AANDENYALAA, which corresponds to the SsrA degradation motif found at the C terminus of abnormal nascent polypeptides in vivo. One SsrA peptide was bound per hexamer of ClpA with an association constant (K(A)) of 5 x 10(6) m(-1). Binding was also assayed by changes in fluorescence of an N-terminal dansylated SsrA peptide, which bound with the same stoichiometry of one per ClpA hexamer (K(A) approximately 1 x 10(7) m(-1)). Similar results were obtained when ATP was substituted for ATPgammaS at 6 degrees C. Two additional peptides, derived from the phage P1 RepA protein and the E. coli HemA protein, which bear different substrate motifs, were competitive inhibitors of SsrA binding and bound to ClpA hexamers with K(A)' > 3 x 10(7) m(-1). DNS-SsrA bound with only slightly reduced affinity to deletion mutants of ClpA missing either the N-terminal domain or the C-terminal nucleotide-binding domain, indicating that the binding site for SsrA lies within the N-terminal nucleotide-binding domain. Because only one protein at a time can be unfolded and translocated by ClpA hexamers, restricting the number of peptides initially bound should avoid nonproductive binding of substrates and aggregation of partially processed proteins.     

Conserved residues in the N-domain of the AAA+ chaperone ClpA regulate substrate recognition and unfolding

Protein degradation in the cytosol of Escherichia coli is carried out by a variety of different proteolytic machines, including ClpAP. The ClpA component is a hexameric AAA+ (ATPase associated with various cellular activities) chaperone that utilizes the energy of ATP to control substrate recognition and unfolding. The precise role of the N-domains of ClpA in this process, however, remains elusive. Here, we have analysed the role of five highly conserved basic residues in the N-domain of ClpA by monitoring the binding, unfolding and degradation of several different substrates, including short unstructured peptides, tagged and untagged proteins. Interestingly, mutation of three of these basic residues within the N-domain of ClpA (H94, R86 and R100) did not alter substrate degradation. In contrast mutation of two conserved arginine residues (R90 and R131), flanking a putative peptide-binding groove within the N-domain of ClpA, specifically compromised the ability of ClpA to unfold and degrade selected substrates but did not prevent substrate recognition, ClpS-mediated substrate delivery or ClpP binding. In contrast, a highly conserved tyrosine residue lining the central pore of the ClpA hexamer was essential for the degradation of all substrate types analysed, including both folded and unstructured proteins. Taken together, these data suggest that ClpA utilizes two structural elements, one in the N-domain and the other in the pore of the hexamer, both of which are required for efficient unfolding of some protein substrates.     

Functional domains of the ClpA and ClpX molecular chaperones identified by limited proteolysis and deletion analysis

Escherichia coli ClpA and ClpX are ATP-dependent protein unfoldases that each interact with the protease, ClpP, to promote specific protein degradation. We have used limited proteolysis and deletion analysis to probe the conformations of ClpA and ClpX and their interactions with ClpP and substrates. ATP gamma S binding stabilized ClpA and ClpX such that that cleavage by lysylendopeptidase C occurred at only two sites. Both proteins were cleaved within in a loop preceding an alpha-helix-rich C-terminal domain. Although the loop varies in size and composition in Clp ATPases, cleavage occurred within and around a conserved triad, IG(F/L). Binding of ClpP blocked this cleavage, and prior cleavage at this site rendered both ClpA and ClpX defective in binding and activating ClpP, suggesting that this site is involved in interactions with ClpP. ClpA was also cut at a site near the junction of the two ATPase domains, whereas the second cleavage site in ClpX lay between its N-terminal and ATPase domains. ClpP did not block cleavage at these other sites. The N-terminal domain of ClpX dissociated upon cleavage, and the remaining ClpXDeltaN remained as a hexamer, associated with ClpP, and expressed ATPase, chaperone, and proteolytic activity. A truncated mutant of ClpA lacking its N-terminal 153 amino acids also formed a hexamer, associated with ClpP, and expressed these activities. We propose that the N-terminal domains of ClpX and ClpA lie on the outside ring surface of the holoenzyme complexes where they contribute to substrate binding or perform a gating function affecting substrate access to other binding sites and that a loop on the opposite face of the ATPase rings stabilizes interactions with ClpP and is involved in promoting ClpP proteolytic activity.     

Turned on for degradation: ATPase-independent degradation by ClpP

Clp is a barrel-shaped hetero-oligomeric ATP-dependent protease comprising a hexameric ATPase (ClpX or ClpA) that unfolds protein substrates and translocates them into the central chamber of the tetradecameric proteolytic component (ClpP) where they are degraded processively to short peptides. Chamber access is controlled by the N-terminal 20 residues (for Escherichia coli) in ClpP that prevent entry of large polypeptides in the absence of the ATPase subunits and ATP hydrolysis. Remarkably, removal of 10–17 residues from the mature N-terminus allows processive degradation of a large model unfolded substrate to short peptides without the ATPase subunit or ATP hydrolysis; removal of 14 residues is maximal for activation. Furthermore, since the product size distribution of Δ14-ClpP is identical to ClpAP and ClpXP, the ATPases do not play an essential role in determining this distribution. Comparison of the structures of Δ14-ClpP and Δ17-ClpP with other published structures shows R15 and S16 are labile and that residue 17 can adopt a range of rotomers to ensure protection of a hydrophobic pocket formed by I19, R24 and F49 and maintain a hydrophilic character of the pore.