Crystal structure of Mpa1-234 revealed tentacle-like coiled-coils
To begin to understand how Pup targets proteins for degradation by the mycobacterial proteasome, we determined the extent of the full-length Mpa coiled-coil by solving the structure of the Mpa1-234 hexamer, which includes the entire coiled-coil and double OB domains (). The crystals were large (0.7 mm), but diffracted poorly (~ 8 Å in the synchrotron beam line, NSLS X29) due to the high solvent content (85%) of the long coiled-coils, an amount that was nearly twice as much as seen in most protein crystals 17
. We improved the diffraction quality by dehydrating the crystals and solved the structure at a resolution of 3.9 Å (, ). The crystals belong to space group P21
with two hexamers per asymmetric unit. The structure was solved by the molecular replacement method using the Mpa double OB fold structure 5
Figure 1 Mpa1-234 hexamer has three 75 Å long coiled-coils needed for Pup recognition. (a) Crystal structure of Mpa1-234 revealed three long coiled-coils formed by six helices that sit atop the hexameric double OB-fold domain. Mpa1-46 was disordered in (more ...)
Data collection and refinement statistics
The N-terminal 51 residues were unstructured, but residues 52-96 formed a contiguous ~75 Å long α-helix in the Mpa crystal. Like ARC and PAN, the six α-helices of the Mpa hexamer formed three pairs of coiled-coils that sat atop three alternating OB domains, thus reducing the six-fold symmetry to three-fold. Strikingly, the coiled-coils protruded like tentacles from the main body of the Mpa hexamer (). The Mpa coiled-coils were in a similar orientation to those in ARC and PAN 10,11
, although the coiled-coils are much shorter in the latter two structures due to truncation or replacement of the coiled-coils in order to facilitate crystallization (Supplementary Fig. 1a,b
The two-stranded parallel coiled-coils in Mpa were formed by one α-helix (trans) that extended to, and dimerized with, its neighboring α-helix (cis) (). The cis α-helix connected to the OB domain via a cis peptide bond between the highly conserved Pro97 and Pro98. The length of the Mpa coiled-coil could accommodate six heptad repeats, but only five were in the structure, leaving a gap between heptads 3 (Leu73) and 4 (Leu87). At the predicted heptad position (residue 80), an alanine took the place of the expected leucine. However, there were three leucines nearby (77, 84, and 85); and Leu85 faced away from the hydrophobic core of the coiled-coil, making it solvent exposed ().
The coiled-coil region of Mpa is needed for Pup recognition
The crystal structure of Mpa1-234 revealed that Mpa1-96 could be further divided into unstructured (Mpa1-45) and coiled-coil (Mpa46-96) domains, thus we wanted to determine if either part were responsible for Pup recognition. We produced Mpa1-46, Mpa46-96, and Mpa1-96 and investigated their binding to various hexahistidine (His6
)-tagged Pup constructs in vitro
. We found that both Mpa1-96 and Mpa 46-96 interacted with Pup whereas Mpa1-46 did not (Supplementary Fig. 2a)
. Taken together, these data indicated that the Mpa coiled-coil was sufficient for the recognition of Pup.
Previous studies have shown that the N-terminal 20 residues of Pup (Pup1-20) are required for in vivo
and in vitro
proteolysis by the mycobacterial proteasome 15,16
. We thus asked if Pup1-20 was required for binding to the Mpa coiled-coil. We produced N-terminal truncated Pup21-64 and full-length Pup, and examined binding with various N-terminal fragments of Mpa in vitro
. We found that Pup21-64 was sufficient to bind to the Mpa coiled-coil (Supplementary Fig. 2b
), thus Pup1-20 is not required for binding to the Mpa coiled-coil, although it is absolutely required for proteasome-mediated proteolysis.
Binding-induced folding of Pup with the Mpa coiled-coil
In order to elucidate how Mpa recognizes Pup, we crystallized Mpa46-96 alone and in complex with Pup21-64, and solved the structures by molecular replacement, with a single α-helix extracted from the Mpa coiled-coil structure (Supplementary Fig. 3
, ; ). In the complex, Mpa46-96 formed a native two-stranded parallel coiled-coil that is essentially the same as observed in Mpa1-234 (). The two helices are designated as Ha and Hb (). Upon binding to the Mpa46-96 coiled-coil, Pup21-64 formed an α-helix (). Pup interacted with the Mpa46-96 coiled-coil with a 1:1 stoichiometry, because both sides of the Mpa46-96 coiled-coil were equivalent and available for Pup binding. In the context of the Mpa hexamer, however, the inner and the outer surfaces of the coiled-coil were not equivalent: the lower part of the coiled-coil at the outside surface was blocked by a crossing loop between β4 and β5 of the first OB domain (, red arrows), thus we predicted that Pup should not be able to bind the outside surface of the coiled-coil in an Mpa hexamer.
The Pup region extending from Ser21 to Ala51 folded into an α-helix, apparently using the Mpa coiled-coil as a template. The C-terminal 13 residues of Pup were disordered in the crystal. The Pup helix interacted in an anti-parallel fashion with the lower half of the Mpa coiled-coil (). The Pup surface interacting with the Mpa coiled-coil was mainly hydrophobic and involved two patches of leucine zipper-like interactions: a smaller patch between Leu32 of Pup and Leu87 and Ala86 of Mpa-Ha; and an extensive patch formed by Leu39, Leu40, Ile43, Val46, and Leu47 of Pup, Ala80, Leu73 of Mpa Ha, and Leu85, Leu84, Ala80, and Leu77 of Mpa Hb ().
Polar residues such as asparagine and aspartate have been shown to drive helix-helix associations through the formation of hydrogen bonds19,20
. A key feature of the Pup and Mpa-coiled-coil interaction is the conserved Asn70 in the Mpa coiled-coil and the Asn50 in Pup: both residues were at the same height in the Mpa-Pup coiled-coil and assumed dual conformations that enabled simultaneous hydrogen bonding within the Mpa coiled-coil, and between Mpa and Pup (). Furthermore, the Pup interaction with the two Mpa helices was asymmetric. Pup contacted one Mpa helix (Ha) extensively via a series of H-bonds among conserved residues, including: Mpa Arg88 with Pup Asp37 (, left); Mpa Asp92 with Pup Thr33 via a water (, left); and Mpa Arg81 with Pup Asp44 and with Asp41 via a water (, center). The Pup interaction with the other Mpa helix (Hb) was weaker: Mpa Arg93 oriented the Glu90 via a H-bond to form two water-mediated H-bonds with Arg28 in Pup (, right).
Pup recognition by the Mpa1-234 hexamer
We next asked if the above-described interaction between the Pup fragment and the Mpa coiled-coil fragment could occur between Pup and Mpa hexamer. We co-crystallized full-length Pup with Mpa1-234. Although the crystals were large, they diffracted X-rays only to a resolution of 4.5 Å in the beam line (X29 of the National Synchrotron Light Source). This was most likely due to high solvent content (81%). In order to prevent the potential phase bias at the coiled-coil region, we solved the Pup:Mpa1-234 complex structure by the molecular replacement method using the Mpa double OB fold structure, which does not contain the coiled-coil region.
In the Pup:Mpa1-234 structure, Pup residues 21-51 formed an α-helix that bound to the lower half of the Mpa coiled-coil in an anti-parallel manner (, ), similar to what was observed in the Pup21-64:Mpa46-96 complex (). Although full-length Pup was used for crystallization, the N-terminal 20 residues were disordered, consistent with our finding that Pup1-20 was not required for binding to the Mpa coiled-coil (Supplementary Fig. 2
). The C-terminal 52-64 residues of Pup were also disordered in the crystal, similar to what we observed in the Pup21-64:Mpa46-96 crystal structure; this unstructured region might serve as a flexible linker between the induced α-helix and the C-terminus that forms an isopeptide bond with a substrate. Pup was not involved in crystalline packing, and binding of Pup did not markedly change the position of the coiled-coils in Mpa. Consistent with our earlier prediction, Pup bound only to the interior side of the coiled-coil in the Mpa hexamer, most likely because the outside surface was partially blocked by cross-barrel loops in Mpa (, red arrows).
Figure 2 Full-length Pup in the context of hexameric Mpa1-234. (a) Overall structure of the Pup:Mpa1-234 complex showing three Pups (red) apparently bound to all three Mpa coiled-coils. The red arrows point to the cross loops of the OB folds, which partially block (more ...)
The electron density of the Pup α-helix was clear but weak, and disappeared at the 3σ threshold, corresponding to approximately a third of the electron density of the Mpa coiled-coil (Supplementary Fig. 4
). While this could be interpreted as disorder in Pup, it is more likely that the weak density was due to Pup binding to only one of the three coiled-coils in any given Mpa hexamer. This binding mode agreed with the one Pup per Mpa hexamer stoichiometry as reported previously 18,21
Alignment of the Mpa coiled-coil in the Pup:Mpa1-234 structure with that in the Pup21-64:Mpa46-96 structure showed that the N-terminal portion of the Pup helix was 3-5 Å farther away from the Mpa coiled-coil in context with the Mpa hexamer (). This was caused by a ~ 4° tilt of the Pup helix around its C-terminus (), which shifted the Pup N-terminus towards the central channel of the Mpa hexamer.
Finally, we observed a large positively charged patch at the middle section of the coiled-coil (, left panel). Positively charged side chains in Mpa were counter-balanced by eight negatively charged residues in Pup (Asp37, Asp38, Asp41, Asp44, Asp45, Glu42, Glu48, and Glu49). At the root of the Mpa coiled-coil, two negatively charged pockets were occupied by Arg28 and Arg29 in Pup (, right panel). Thus, electrostatic interactions also appeared to play a role in the recognition of Pup by Mpa.
Pup-Mpa interacting residues are critical for proteolysis
The presence of negatively charged pockets in the Mpa coiled-coil that seemed to make robust interactions with Pup Arg28 and Arg29 () prompted us to test the importance of the positively charged residues in protein degradation. Additionally, based on the crystal structure of Pup21-64:Mpa46-96 (), several conserved hydrophobic residues within the α-helical region of Pup appeared to be important for interacting with Mpa. We introduced three pairs of double mutations into a Pup reporter fusion construct (Pup-Zur-His6
: one pair mutated arginines 28 and 29 to alanine, and two pairs disrupted hydrophobic regions of Pup (L39S L40S and V46S L47S) ( and ). Strikingly, all three double mutations in Pup nearly or completely abolished degradation of the reporter in M. smegmatis
(Msm) (). This result demonstrated the essentiality of the Pup helical region for the recognition of Pup by Mpa for the proteolysis of a substrate by the mycobacterial proteasome.
Figure 3 Essentiality of the Pup helical region for proteasomal degradation supports a binding-induced folding recognition mechanism by Mpa. (a) Site-directed mutations in Pup resulted in abrogated degradation of a Pup-linear fusion by the Msm proteasome. The (more ...)