I27₂-PimA-I27₂ Polyprotein Production

The polyprotein I27₂-PimA-I27₂ was generated by using recombinant DNA technology based on an iterative process of digestion and ligation of DNA fragments as described previously (28). The pimA gene from M. smegmatis was amplified by standard PCR using oligonucleotide primers pimA_BamHI_Fwd (5′-CGCGGATCCGGCTCTGGCGCGATGCGTATCGGGATGGTCTGCC-3′) and pimA_SacI_Rev (5′-ATAATAGAGCTCTTAGCAACAAGATCTGACCGATTCTCCGGCCGTCTG-3′), Phusion DNA polymerase (Finnzymes, Thermo Fisher Scientific), and the plasmid pET29a-MspimA as the DNA template (27). The PCR fragment was digested with BamHI and SacI and ligated to the vector pT7-I27₂ harboring two consecutive copies of the immunoglobulin (Ig) domain of the giant muscle protein titin, I27. The resulting plasmid, pT7-I27₂-PimA, was further digested with BglII and SacI, and the fragment LF-pT7-I27₂-PimA was purified. The pT7-I27₂ was digested with BamHI and SacI, and the fragment SF-I27₂ was purified. Both fragments LF-pT7-I27₂-PimA and SF-I27₂ were ligated, and the resulting construct was named pT7-I27₂-PimA-I27₂. Digestion of pT7-I27₂-PimA-I27₂ with BamHI and SacI allowed in-frame cloning of the I27₂-PimA-I27₂ into the expression vector pQE80L (Qiagen, Chatsworth, CA). As depicted in supplemental Fig. 1S, the full-length construct resulted in the following amino acid additions. (i) The N-terminal sequence is Met-Arg-Gly-Ser-His₆-Gly-Ser-I27. (ii) The junction between the I27 domains (BamHI-BglII hybrid site) is Arg-Ser. (iii) The junction between the second I27 domain and PimA is Arg-Ser-Gly-Ser-Gly-Ala. (iv) The junction between PimA and the third I27 domain is Arg-Ser. In addition, the resulting construct contained two Cys residues at the C terminus, allowing the covalent attachment of the polyprotein to the gold-coated coverslips. E. coli BL3 cells transformed with pQE80L-I27₂-PimA-I27₂ were grown in 400 ml of LB medium supplemented with 30 μg/ml kanamycin and 50 μg/ml carbenicillin at 37 °C. When the culture reached an A₆₀₀ value of 0.6, I27₂-PimA-I27₂ expression was induced by adding 1 mm isopropyl β-d-1-thiogalactopyranoside (MIP). After overnight incubation at 25 °C, cells were harvested and resuspended in 16 ml of 50 mm Tris-HCl, pH 8.0 (solution A) containing protease inhibitors (Complete EDTA-free, Roche Applied Science). Cells were then disrupted by sonication (five cycles of 1 min each), and the suspension was centrifuged for 20 min at 10,000 × g. The supernatant was applied to a Talon resin column (Clontech) followed by a gel filtration step in a Superdex 200 column (GE Healthcare) equilibrated in 10 mm Hepes, pH 7.2, 1 mm EDTA, 150 mm NaCl (solution B). The resulting I27₂-PimA-I27₂ preparation displayed a single protein band when run on a 12% NuPAGE® Bis-Tris precast gel stained with SimplyBlue^TM SafeStain (Invitrogen). The purified I27₂-PimA-I27₂ polyprotein was stored at −80 °C for further use in single molecule experiments.

I27-PimA Protein Production

The pimA gene from M. smegmatis was amplified as described for the I27₂-PimA-I27₂ construct. The PCR fragment was digested with BamHI and SacI and then ligated to the vector pT7-I27 harboring one copy of the I27 module. The resulting plasmid, pT7-I27-PimA, was further digested with BamHI and SacI and ligated to the expression vector pQE80L (Qiagen). As depicted in supplemental Fig. 2S, the full-length construct resulted in the following amino acid additions. (i) The N-terminal sequence is Met-Arg-Gly-Ser-His₆-Gly-Ser-I27. (ii) The junction between the I27 domain and PimA is Arg-Ser-Gly-Ser-Gly-Ala. In addition, the final construct contained two Cys residues at the C terminus. E. coli DH5α cells (Invitrogen) transformed with pQE80L-I27-PimA were grown in 3000 ml of 2×YT medium supplemented with 50 μg/ml carbenicillin at 37 °C. When the culture reached an A₆₀₀ value of 0.6, I27-PimA expression was induced by adding 1 mm isopropyl β-d-1-thiogalactopyranoside (MIP). After overnight incubation at 18 °C, cells were harvested and resuspended in 40 ml of solution A containing protease inhibitors (Complete EDTA-free, Roche Applied Science). Cells were then disrupted by sonication (five cycles of 1 min each), and the suspension was centrifuged for 20 min at 10,000 × g. The supernatant was applied onto a HisTrap column (GE Healthcare) followed by a gel filtration step in a Superdex 75 column (GE Healthcare) equilibrated in buffer 50 mm Tris-HCl, pH 7.5, 150 mm NaCl (solution C). The resulting I27-PimA preparation displayed a single protein band when run on a 12% NuPAGE Bis-Tris precast gel stained with SimplyBlue SafeStain (Invitrogen). The purified I27-PimA fusion protein was stored at 4 °C and then concentrated for small angle x-ray scattering (SAXS) measurements by using a Vivaspin 20 spin concentrator (Vivascience) with a 10-kDa-molecular mass cutoff. The homogeneity of I27-PimA was confirmed by dynamic light scattering (data not shown).

Single Molecule Force Spectroscopy Measurements

5–15 μl of I27₂-PimA-I27₂ from the most concentrated fraction obtained after gel filtration were deposited onto an evaporated gold coverslip. Veeco silicon nitride MLCT (Bruker, Camarillo, CA) cantilevers were mounted in a fluid cell carrier. The laser beam was centered at the tip of the cantilever and carefully calibrated. Spring constants around 15 piconewtons/nm were obtained using the equipartition theorem (29). The cantilever was kept in contact with the gold coverslip for 1–2 s before retraction. We used a custom-built atomic force microscopy setup that allows a strict control of the force based on an electronic feedback. Experiments were performed at room temperature in 10 mm Hepes, pH 7.2, 1 mm EDTA, 150 mm NaCl. Only traces displaying four steps corresponding to I27 unfolding were considered. Because of the low stability of the construct I27₂-PimA-I27₂, single molecule experiments were performed within 3–4 days after purification. During that time, the purified I27₂-PimA-I27₂ polyprotein construct was demonstrated to conserve full enzymatic activity in vitro (Ref. 8 and data not shown). To calculate the contour length of the pulled polypeptide, we applied the wormlike chain model with a persistent length of 0.4 nm. Hence, the wormlike chain curves in Fig. 3 correspond to the theoretical extension of the N- and C-terminal Rossmann fold domains, 163 and 119 amino acids, respectively, and I27. For a more accurate estimation of the contour length, the length of the folded N- and C-terminal Rossmann fold domains (1.8 and 2.3 nm, respectively) was subtracted. The wormlike chain was fitted in force-extension curves as well to estimate the initial extension before the rupture of the first I27 domain (30).

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FIGURE 3.

Extension of I27₂-PimA-I27₂ during a linear increase in force. A and B, typical force-ramp traces of I27₂-PimA-I27₂ in the absence and presence of GDP, respectively. Step size events of PimA are annotated in red. C and D, distribution of step sizes and unfolding forces of I27 and PimA unfolding events from 50 unambiguous traces. Theoretical extensions following the wormlike chain of I27 and the N-terminal and C-terminal β-sheets of PimA are plotted in blue, red dashed, and red dotted lines, respectively. pN, piconewtons.

Small Angle X-ray Scattering Measurements

Synchrotron x-ray diffraction data for recombinant purified MsPimA apo and MsPimA in the presence of GDP (27) were collected on a pixel Pilatus 1M detector at the European Molecular Biology Laboratory X33 beamline (Deutsches Elektronen-Synchrotron, Hamburg, Germany; Refs. 31 and 32), whereas data for the fusion I27-PimA protein were collected at the European Molecular Biology Laboratory P12 beamline (Deutsches Elektronen-Synchrotron) on a pixel Pilatus 2M detector. The scattering patterns were measured with a 2-min exposure time for protein samples at a minimum of three different protein concentrations ranging from 1 to 10 mg/ml at the X33 and with a 1-s exposure time at the P12. Samples were incubated with 2 mm dithiothreitol before data collection. To check for radiation damage, four 30-s exposures were compared at the X33, and 20 50-millisecond exposures were compared at the P12; no radiation damage was observed. Using the sample-to-detector distance of 2.7 m (3.1 m at the P12), the range of momentum transfer values is 0.009 < q < 0.5 Å⁻¹ at the X33 and 0.008 < q < 0.47 Å⁻¹ at the P12 (q = 4π sin(θ)/λ where 2θ is the scattering angle and λ = 1.5 Å (1.24 Å for the P12 beamline) is the x-ray wavelength). Data were processed using standard procedures and extrapolated to infinite dilution by the program package PRIMUS (33). The forward scattering (I(0)) was evaluated using the Guinier approximation (34) assuming the intensity is represented as I(q) = I(0)exp(−(qR_g)²/3) for a very small range of momentum transfer values (q < 1.3/R_g). The maximum dimensions (D_max), the interatomic distance distribution functions (P(r)), and the radii of gyration (R_g) were computed using GNOM (35). The molecular mass of the protein was evaluated by comparison of the forward scattering with that from a reference solution of bovine serum albumin (66 kDa).

Ab Initio Shape Determination

The low resolution structures of MsPimA apo and MsPimA·GDP complex were calculated ab initio by using the program DAMMIF (36). Structure clustering and averaging were carried out with the program DAMCLUST (37). The results and statistics are summarized in Table 1. For each data set, pairwise alignment of structures was carried out, and the normalized spatial discrepancy (NSD) was calculated (38). The clustering process groups structures with lower NSD. In general, NSD values close to 1 indicate that the two structures are similar. Structures that deviate by more than 2 are taken as outliers and removed. For all calculations, three outlier models were rejected. Average NSD values within clusters are between 0.5–7, indicating a very good structural agreement for each cluster. Furthermore, average NSD values for structures of different clusters are between 0.7 and 1.0, indicating similarity between clusters.

TABLE 1

Parameters of the unliganded and GDP-bound forms of PimA and the I27-PimA fusion protein calculated from the SAXS data

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^a Values in parentheses are estimated errors approximated to the last decimal place.

^b q-range used for calculation of P(r) function.

^c R_g value extrapolated to 0 mg/ml concentration.

^d Molecular mass estimated by calculation of I(0) and comparison against bovine serum albumin.

^e Values in parentheses are standard deviations calculated for 20 ab initio calculations.

^f MONSA R-factors are reported for each experimental curve employed in the calculation (i.e. SAXS data of I27-PimA and PimA apo). NSD values are reported for the individual phases reconstructed (i.e. I27 and PimA apo).

The ab initio reconstruction of the fusion protein I27-PimA was carried out by MONSA. MONSA is based on DAMMIN and allows bead modeling of macromolecular complexes by simultaneously fitting multiple experimental data (39, 40). The search volume was defined as a sphere of radius equal to half the maximum dimension of I27-PimA (D_max = 120 Å) as computed by GNOM. Within the search volume, a grid of points is defined with a constant distance of 2 Å between points, resulting in dense packing of spheres. Each sphere within the search volume might correspond to one of the following components: solvent, I27, or PimA, represented by an integer number, 0, 1, or 2. To facilitate computation and to keep physical sense with respect to the experimental data (i.e. low resolution of data and connectivity of each component), a number of conditions are applied during structural calculations accounting for the likely connectivity of two arbitrarily selected atoms belonging to a given component and its compactness. The minimization procedure fits the two experimental data sets, PimA and I27-PimA, to the corresponding calculated data obtained from computed structures. It is carried out by simulated annealing (41) starting from a random configuration X₀ where the assignment of a single bead to a given component is changed randomly (a move from X to X′). In the case that the change produces an improvement of fitness (Δ = f(X′) − f(X) < 0), it is accepted. Otherwise, it can still be accepted with a probability of exp(−Δ/T) where T is the annealing temperature, which decreases as the calculation progresses toward a minimum. The described approach allowed us to unambiguously differentiate the relative location of I27 and PimA within the fusion protein and to correctly orientate the N- and C-terminal domains of PimA as observed in the crystal structure in the solution structures obtained by SAXS techniques.

PimA Unfolds following Heterogeneous Multiple Step Mechanical Unfolding Pathways

To further characterize PimA unfolding, we used a force ramp protocol with a linearly increasing force (51). This protocol explores a wide range of forces and is therefore more suitable to examine and segregate different unfolding pathways. We increased the force from 0 to 250 piconewtons over a time period of 5 s, obtaining 50 well defined traces in the presence and absence of GDP. Extension of I27₂-PimA-I27₂ was identified by the presence of four uniform I27 unfolding steps (Fig. 3, A and B, and Ref. 50). For each distinguishable step appearing in the force ramp, the step size and the force at which the event occurs are reported in the scatter plots in Fig. 3. Remarkably, PimA unfolds following multiple steps at low force. The steps observed during the ramp are well defined and can be characterized by its size and force. However, the large dispersion in step sizes and unfolding forces for PimA unfolding reflects the heterogeneity of mechanical unfolding pathways, analogous to the mechanical unfolding of molten globule states (52, 53). Similar results were obtained in the presence of 5 mm GDP (dissociation constant K_d of 0.03 μm (ΔH = −14.0 kcal·mol⁻¹; Fig. 2), which is a well known thermal stabilizer of the enzyme. GDP binds tightly to the C-terminal domain through a network of hydrogen bonds and stabilizes the enzyme by coupling the two N- and C-terminal lobes in a closed state (27).

Fig. 4 reveals a particularly well resolved trace illustrating a possible pathway in which the theoretical extension coincides with the one-step rupture of the 163 amino acids forming the N-terminal β-sheet and the 119 amino acids forming the C-terminal β-sheet (Fig. 4A). We propose that the first four shorter steps bear out the detachment of the C-terminal helices (Fig. 4B, yellow). Then the linker and the N-terminal domain unfold in a two-step manner. The last step is compatible with the full extension of the C-terminal β-sheet. This single trace depicts one of the multiple routes that are available to fully extend PimA under force. Step sizes larger than the theoretical unfolding length of the two β-sheets would suggest the co-rupture of larger structural elements.

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FIGURE 4.

A single unfolding pathway of I27₂-PimA-I27₂. A, a well resolved trace illustrating a PimA unfolding pathway. The force applied increases from 0 to 250 piconewtons (pN) in 5 s. The first steps describe extensions of 6, 4, 6, 6, 22, 35, and 42 nm as the force increases, revealing intermediates along the unfolding pathway of PimA. The 42-nm step, colored in red, correlates with the unfolding of the C-terminal Rossmann fold. The four 24-nm steps occurring at higher force are colored in blue and correspond to I27 unfolding. Gray arrows display the direction of the force applied to PimA and I27 in their native states. B, topology of the proposed intermediates after unraveling the hinge (α7) and the last three α-helices (α12, α13, and α14) of PimA. The N-terminal domain unfolds first followed by the C-terminal domain. In contrast to PimA, I27 unfolds in an all-or-none unfolding pathway.

The mechanical properties of proteins are strongly influenced by their structural elements (28). To date, the strongest mechanical element corresponds to the β-sandwich topology characteristic of Ig and Ig-like proteins (54). As depicted in Figs. 3 and ​and4,4, I27 unfolds in an all-or-none unfolding pathway identically to proteins with similar topology such as ubiquitin and protein L (51, 55). In all these proteins, two parallel β-strands linking the N and C termini firmly protect the majority of native contacts before unraveling the rest of the protein. As soon as the parallel β-strand interactions break, the remaining structural elements unfold without any force resistance. Indeed, mutations in this structural motif in Ig or Ig-like proteins markedly reduce mechanostability (56). Interestingly, different unfolding forces have been reported for homologous Ig domains. The presence of parallel β-strands motifs does not fully explain the mechanostability of I27, and other properties have to be considered (48).

PimA topology is remarkably more complex than that of I27. PimA shares with mechanically stable proteins the presence of β-sheets in both Rossmann fold domains (14) with numerous parallel β-strands (Figs. 1 and ​and4),4), suggesting a strong mechanical stability. The N-terminal end folds into a β-strand (β1; residues 1–7) and makes strong interactions with β2 and β4 strands of the central β-sheet core of the first domain. In contrast, the C-terminal region terminates with the classical GT-B pattern of three long α-helices, α12 (residues 315–328), α13 (residues 330–347), and α14 (residues 349–363), with α14 crossing over to the N-terminal domain (Figs. 1 and ​and4).4). This hinge region is completed by residues 165–190, which include α7. Because α-helices are mechanically weak elements (57, 58), all helices are expected to unfold first at low force, allowing PimA to reach a conformation where the two Rossmann fold domains are separated. In this scenario, the GDP binding site is dislocated very early in the unfolding pathway. Therefore, when the force is applied to a PimA·GDP complex, the affinity for GDP is expected to drop following the mechanical disruption of the binding site. This model agrees with the observation that mechanical unfolding of PimA in the presence or absence of GDP gives rise to similar distributions of events (Fig. 3B).

Once the hinge has extended, parallel β-strands in both Rossmann fold domains are exposed to force (Fig. 4B). This situation induced by force is equivalent to native mechanostable proteins such as I27. However, contrary to I27, PimA unfolds with a remarkably wide distribution of step sizes at low force where no common structure dominates. Our results suggest that the presence of parallel β-strands is not sufficient to explain the mechanical properties of PimA where the presence of marginal stable structural elements within the protein allows the Rossmann fold domains to unfold along a variety of pathways. We propose that the partial unfolding of helical regions and the hinge at low force leads to a marked perturbation of the PimA energy landscape where a significant number of native contacts are disrupted before the two β-sheets are under force.

The Open and Closed States of PimA as Visualized by SAXS

To gain insight into the conformational changes that occur in PimA, the solution structures of the enzyme in its unliganded and GDP-bound forms were solved by SAXS. SAXS proved to be a powerful technique capable of providing structural information of flexible and dynamic proteins in solution (59). The interatomic distance distribution function (P(r)) computed for PimA apo and PimA in the presence of GDP is shown in Fig. 5. The molecular mass determined from the scattering data confirmed that the protein is monomeric in solution in agreement with size exclusion chromatography and analytical ultracentrifugation experiments (see “Experimental Procedures,” Table 1, and Refs. 14 and 27). It is worth noting that the protein remained in a monomeric state even in the presence of GDP.

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FIGURE 5.

Solution SAXS data statistics. A, scattering curves of PimA apo, PimA·GDP complex, and I27-PimA fusion polyprotein. B, P(r) function distributions of PimA apo, PimA·GDP complex, and I27-PimA fusion polyprotein. C, plot of R_g as a function of protein concentration. SAXS measurements were performed at concentrations of 2.2, 3.5, and 5.5 mg/ml PimA. R_g values at a concentration of 0 mg/ml of 28.5 and 27.5 Å were calculated for PimA apo and PimA·GDP complex, respectively.

The R_g values obtained for PimA apo and the PimA·GDP complex revealed a reduction in R_g (ΔR_g) of −1.0 Å, indicating that GDP binding leads to a compaction of PimA, clearly supporting the existence of the open and closed states of the enzyme (Table 1 and Ref. 27). Several lines of experimental evidence provide strong confidence in this model. The relevance of the β-PO₄ in the stabilization of the closed conformation of PimA was first studied by limited proteolysis (27). PimA was rapidly degraded after incubation with elastase. N-terminal sequencing of the two predominant species of 23 and 15 kDa revealed two exposed sites located in α9 and the connecting loop β7-β8 at the junction between N- and C-terminal domains. The α9 contains two important residues involved in GDP-Manp recognition: Asp-253 and Lys-256. Interestingly, when PimA was incubated in the presence of GDP, the enzyme was protected from the action of elastase even after 90 min, suggesting a conformational rearrangement. A close inspection of the crystal structure of the PimA·GDP complex revealed that the enzyme crystallizes in a closed conformation with the active site buried between both Rossmann fold domains (14). Two residues of the N-terminal domain were found to interact with GDP: Pro-14 in the connecting loop β1-α1 stabilizes the guanine heterocycle by a van der Waals stacking interaction, whereas Gly-16 at the top of α1 hydrogen bonds the β-PO₄ of GDP (14). Moreover, Arg-196 and Lys-202, largely conserved in PimA orthologues but located in the C-terminal domain, make electrostatic interactions with the β-PO₄, thus restricting its position into the catalytic site. Importantly, the α-PO₄ of GDP does not interact with any particular residue from the enzyme (14). Structural comparisons of the unliganded and nucleotide-diphospho-sugar-bound forms of MshA from Corynebacterium glutamicum (CgMshA) and the glycogen synthase from Agrobacterium tumefaciens (AtGS) and E. coli (EcGS) revealed that a substantial subdomain rotation is required to achieve the closed state (20, 21, 23). The residues Gly-23, Arg-231, and Lys-236 in CgMshA; Gly-18, Arg-299, and Lys-304 in AtGS; and Gly-18, Arg-300, and Lys-305 in EcGS interact with the β-PO₄ of uridine 5-diphosphate (UDP) and adenosine 5-diphosphate (ADP), respectively, and are structurally equivalent to Gly-16, Arg-196, and Lys-202 in the closed conformation of MsPimA.

The “open-to-closed” motion has also been monitored in the absence and presence of GDP and guanosine by sedimentation velocity analytical ultracentrifugation studies on pure MsPimA (27). The unliganded form of the enzyme sedimented as a single homogeneous species with an average sedimentation coefficient of 3.22 S, which is consistent with an asymmetric monomeric protein. Upon the addition of equimolar GDP, the sedimentation coefficient increased to 3.53 S, indicating the formation of a more symmetrical and compact structure. The addition of guanosine in which the α-PO₄ and β-PO₄ are missing did not significantly affect the sedimentation coefficient value of the mannosyltransferase, supporting the requirement of the β-PO₄ to trigger the closed conformation. Moreover, isothermal titration calorimetry measurements revealed that guanosine bound to PimA with a binding constant ∼10³-fold smaller than that of GDP. The GDP binding also produced a stabilizing effect on MsPimA characterized by an increment of 3.5 °C in the melting temperature value as observed by differential scanning calorimetry and circular dichroism (27). In summary, GDP induces the closing movement of PimA with a clear increment of the sedimentation coefficient value that is correlated with a reduction of the radii of gyration and accompanied by a marked stabilization of the enzyme.

Conformational Flexibility of PimA

The ab initio low resolution calculated envelopes for PimA apo and PimA·GDP complex displayed the characteristic two-domain organization of the GT-B enzymes with the deep fissure between the N- and C-terminal lobes including the catalytic center (see “Experimental Procedures,” Table 1, and Figs. 6 and ​and7).7). However, a comparison of the crystal structure of PimA·GDP complex with the solution structures obtained by SAXS revealed significant differences in the conformation of one of the Rossmann fold domains of the protein. To unambiguously identify the N- and C-terminal domains in the ab initio models, SAXS data were collected on a I27-PimA polyprotein where the 10-kDa I27 module was fused to the N-terminal end of MsPimA (see Table 1 and “Experimental Procedures” for details). The experimental data clearly demonstrate the position of the I27 protein in close proximity to a protruding lobe on the N-terminal domain (Table 1 and Fig. 6). It is worth noting that the different structures depicted in Fig. 7 do not directly prove the flexibility of PimA but rather stem from the potential ambiguity of ab initio reconstructions in SAXS. Nevertheless, they provide insight into the possible structural flexibility by depicting possible protein conformations compatible with the scattering data.

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FIGURE 6.

Low resolution models of PimA in solution. Left, average low resolution structure of PimA apo with the high resolution crystal structure of PimA·GDP complex (Protein Data Bank code 2GEK) fitted by rigid body docking. Middle, average low resolution structure of PimA·GDP complex with the high resolution crystal structure of PimA·GDP complex fitted by rigid body docking. Right, average low resolution structure of I27-PimA fusion polyprotein with the high resolution crystal structures of I27 and PimA·GDP complex fitted by rigid body docking.

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FIGURE 7.

Gallery of selected PimA ab initio SAXS models. Individual ab initio reconstruction runs of PimA apo (left) and PimA·GDP complex (right) showing structural variability are shown.

The N-terminal domain of PimA has been proved to participate in acceptor recognition involving important conformational changes in the protein (14, 27). Specifically, when incubated with PI, both the unliganded and the GDP-bound forms of the enzyme became highly sensitive to elastase treatment, indicating that PI triggers a significant conformational change able to modify the closed GDP-induced conformation. Furthermore, analytical ultracentrifugation experiments demonstrated that the addition of PI to the enzyme resulted in a significant change in the sedimentation coefficient values of both PimA apo and PimA·GDP complex consistent with the formation of less compact structures, which correlates with a reduction of the melting temperature by 1.5 and 0.4 °C, respectively. The crystal structure of the PimA·GDP complex displayed two regions on the N-terminal domain comprising residues 118–140 and 149–165 with particularly high B-factor values suggesting structural flexibility. These residues are located in three α-helices, α4, α5, and α6, and primarily define a hydrophobic tunnel, which is compatible with the binding of fatty acyl chains of PI or phospholipids of the mycobacterial plasma membrane. Thus, this particular region of PimA could certainly experience conformational flexibility and might explain the presence of the extra lobe in the SAXS structure.

The conformation of membrane-associated proteins can certainly be affected by binding to the lipid bilayer, thus modulating protein function. These conformational changes can be quite different in nature, ranging from subtle modifications such as the reorientation of lateral chain residues to more complex structural rearrangements (60). For example, the orientation of transmembrane domains in polytopic integral membrane-associated proteins has been proved to be dependent on membrane phospholipid composition during initial assembly as well as on changes in lipid composition postassembly (61). A dramatic conformational change has also been reported for the pore-forming toxin pneumolysin where two of the four domains refold along with the deformation of the membrane (62). Interestingly, recent studies suggest that the molten globule states play an important role in cellular processes that involve membrane interaction. The translocation of the N-terminal domain of the anthrax lethal factor across the membrane requires mechanical unfolding of a molten globule-like state (63). Moreover, the interaction of the peripheral membrane-associated α-lactalbumin to phospholipid bilayers of various compositions revealed the membrane-bound state to resemble a molten globule-like state (64). It is worth noting that the amphipathic helix C is a critical determinant for the adsorption and further integration of the α-lactalbumin into the membrane. In that sense, PimA displays an amphipathic helix on its N-terminal domain that has been proved to be essential for membrane binding and activity in vitro and in vivo (27). The occurrence of metastable states of PimA might be reflective of conformational variability of the enzyme restricted to the N-terminal domain and the association to the acceptor substrate and/or lipid bilayer. However, further studies will be required to define in detail the residues responsible for the observed structural variability.