Mechanism of glycogen synthase inactivation and interaction with glycogenin

Glycogen is the major glucose reserve in eukaryotes, and defects in glycogen metabolism and structure lead to disease. Glycogenesis involves interaction of glycogenin (GN) with glycogen synthase (GS), where GS is activated by glucose-6-phosphate (G6P) and inactivated by phosphorylation. We describe the 2.6 Å resolution cryo-EM structure of phosphorylated human GS revealing an autoinhibited GS tetramer flanked by two GN dimers. Phosphorylated N- and C-termini from two GS protomers converge near the G6P-binding pocket and buttress against GS regulatory helices. This keeps GS in an inactive conformation mediated by phospho-Ser641 interactions with a composite “arginine cradle”. Structure-guided mutagenesis perturbing interactions with phosphorylated tails led to increased basal/unstimulated GS activity. We propose that multivalent phosphorylation supports GS autoinhibition through interactions from a dynamic “spike” region, allowing a tuneable rheostat for regulating GS activity. This work therefore provides insights into glycogen synthesis regulation and facilitates studies of glycogen-related diseases.

Analysis of the GS-GN(WT) sample identified a species with an average molecular weight of 139 534 kDa and the measured peak was broader than the non-glucosylated species (Fig. 1f).

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While mass photometry measurements lack the resolution to ascertain the precise molecular 141 mass of heterogeneously glucosylated species, the observed increase in average molecular 142 mass and overall distribution of the WT complex when compared to the Y195F complex is 143 consistent with the observed higher molecular weight of WT GN1 glucosylated species (Fig.   144 1d and Supplementary Fig. 3b). To understand how GS and GN interact and to reveal the overall shape of the GS-GN complex 147 we performed negative stain electron microscopy (nsEM) of the WT and Y195F complexes. 148 2D class averages show two GN dimers, one on either side of a GS tetramer, for both WT and 149 mutant complexes (Fig. 1g). Final 3D maps for both complexes are consistent with the 2D 150 classes, and the reconstructed 3D EM density map can accommodate a GS tetramer flanked 151 by two GN dimers (Fig. 1h). This nsEM confirms a 4:4 stoichiometry and is consistent with 152 previous findings showing that GS can interact with four GN C-terminal peptides 153 simultaneously 4,5,17 . Surprisingly, GN dimers do not engage the GS tetramer in an identical 154 fashion, with one GN dimer tilted slightly towards GS and bringing it closer to one of the GS 155 subunits (Fig. 1h). Collectively, these results provide the first glimpse of the glycogen initiating 156 particle, where two GN dimers can engage a single GS tetramer.

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Phosphorylated human GS is in the inactive state 159 160 GS is regulated by both allosteric activation by G6P and inhibition via phosphorylation of its 161 N-and C-terminal tails 2 (Fig. 1c). Mechanistic and structural studies of yeast GS have 162 elegantly dissected its allosteric activation by G6P 30,34 . However, GS structures to date were 163 from protein preparations produced in bacterial expression systems and thus could not provide 164 insights into the phospho-regulatory apparatus. Our GS-GN preparations are from eukaryotic 165 expression systems and therefore provide an opportunity to study the inactive GS form. We 166 confirmed that GS was phosphorylated at sites 2 (S8) and 3a (S641) and the enzyme 167 preparation was inactive unless stimulated by G6P or dephosphorylation ( Fig. 2a and 2b).

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Protein phosphatase 1 (PP1) and lambda protein phosphatase (lambda PP) treatment 169 resulted in faster migration of GS by SDS-PAGE and also a reduction in signal detected by 170 specific phosphorylation site antibodies ( Fig. 2a and Supplementary Fig. 4a). Notably, we 171 see only minor dephosphorylation of the GS-GN(Y195F) complex with PP1 alone, which was 172 associated with a 5-fold increase in basal activity (-G6P) ( Fig. 2a and 2b). We observed a 15-173 fold increase in basal activity when GS is dephosphorylated by both PP1 and lambda PP (Fig.   174 2b). The phosphorylated and dephosphorylated GS forms were similarly active after addition 175 of G6P (Fig. 2b), which is consistent with studies using GS from endogenous sources 39,40 . 8 Fig. 2 Cryo-EM structure of human GS-GN 34 complex a Immunoblot for the indicated human GS phosphorylation sites and total GS. b Activity of GS-GN(Y195F) with and without the addition of lambda protein phosphatase (lambda PP) and protein phosphatase 1 (PP1) (left) and -/+ G6P activity ratio (right). Upon G6P saturation, GS reaches similar activity levels regardless of phosphorylation state. Data are mean +/-S.E.M. from n=2 and representative of two independent experiments. One-way analysis of variance (Tukey's post hoc test); §= p<0.05, +PP1 vs. +PP1+lambda PP (-G6P) (left). *= -PP1-lambda PP vs. +PP1 vs. +PP1+lambda PP, §= +PP1 vs. +PP1+lambda PP (right). c 2.6 Å cryo-EM map of the GS tetramer coloured by corresponding chain. Density corresponding to the GN 34 C-terminal region is shown in green. d Human GS-GN 34 cartoon model shown in ribbons coloured by corresponding chain (left). Interaction between GS and GN 34 (right). e Unsharpened cryo-EM map shown at a lower threshold to visualise the "spike" region depicted in grey (left). The N-and C-terminal tails of two protomers converge and form the "spike" region (right).
To better understand the extent of phosphorylation we used tandem mass spectrometry (MS/MS) after proteolysis with either trypsin, chymotrypsin or elastase to map the phosphorylation sites of human GS. This resulted in a sequence coverage of 97%, which is 180 higher than the 73% 35 and 65% 36 sequence coverage achieved in previous studies (Table 1 181 and Supplementary Fig. 4b). Our analysis identified canonical sites 2, 3a, 3b, 4 and 5 (S8, 182 S641, S645, S653, S657), and also non-conventional sites (S412, S652, S727, S731). In 183 addition, we could detect human GS site 2 (S8) phosphorylation by mass spectrometry for the 184 first time in a recombinant enzyme preparation. Together, these results show that expression 185 in insect cells is sufficient to achieve phosphorylation at multiple inhibitory sites and to provide 186 suitable enzyme preparations to study inactive GS. 187 188  Fig. 1g and 1h) Fig. 5a) and subsequent 3D maps. Although we could detect the presence 203 of GN after data processing without the application of symmetry averaging (Supplementary 204 Fig. 6c), it was not possible to trace the connecting residues between the GN globular domain 205 and the C-terminal GN 34 region that binds GS. To gain a higher resolution structure for the 206 human GS, we applied D2 symmetry and achieved a global resolution of 2.6 Å (EMDB-14587) 207 ( Fig. 2c, Supplementary Fig. 5 and Supplementary Table 1). The 3D reconstruction 208 revealed a tetrameric arrangement of human GS in agreement with the crystal structures of 209 the C. elegans GS and yeast GS enzymes, with root mean square deviation (RMSD) values 210 of 1.1 Å (between 484 Ca atom pairs) and 0.9 Å (between 522 Ca atom pairs) respectively 211 ( Fig. 2d, Supplementary Fig. 7a and 7b).

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Density for the C-terminal GS interacting region of GN allows for model building of residues 217 300-332 (human GN 34 ). Four GN peptides bind to the GS tetramer, and these residues form 218 a helix-turn helix, where the first helix is denoted binding helix 1 (BH1) and the second as BH2 219 (Fig. 2d). This is consistent with the C. elegans GS-GN 34 crystal structure 22 , with an RMSD 220 value of 0.8 Å (between 30 Ca atom pairs) (Supplementary Fig. 7c). The interaction interface 221 between human GS, namely a4, a9 and a10, and human GN 34 is mediated by a combination 222 of hydrophobic and hydrogen bonding interactions and is consistent with the interactions 223 observed for GS-GN 34 from C. elegans 22 (Fig. 2d and Supplementary Fig. 7c).

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A unique feature of metazoan GS is that both N-and C-terminal tails are phosphorylated, but 228 the mechanism by which they participate in enzyme inactivation has remained elusive. We 229 were able to build a model for the N-terminus starting from residue 13, and of the C-terminus 230 up to residue 625, and then from 630-639 (chain A/C) and 630-642 (chain B/D), that could 231 help understand the mechanisms of GS inactivation (PDB 7ZBN). The N-and C-terminal tails 232 of each GS protomer lie almost parallel to each other, and travel side by side along the GS 233 tetrameric core to reach the centre (Fig. 3a, right panels). Here, the C-terminal tail (chain A) 234 meets the C-terminal tail from an adjacent GS protomer (chain B), which has travelled from 235 the opposite direction (Fig. 3a, right panels). A 2.8 Å cryo-EM map of GS generated without 236 the application of D2 symmetry averaging (EMDB-14587) (Supplementary Fig. 6a and 6b),

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suggests that one C-terminal tail disengages with the GS core earlier than the other C-terminal tail from the adjacent chain. The C-terminal tail from chain B continues to travel further across 239 the regulatory helices than chain A, prior to traversing away from the core (Fig. 3a). This 240 allows chain B to engage with the regulatory helices a22, specifically phosphorylated S641 241 interacting with residues R588 and R591, which come from two GS protomers to form a 242 positively charged pocket we have termed the "arginine cradle" (Fig. 3a

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Notably, the EM structures maps show density for an inter-subunit region that extends from 268 the N-and C-termini of two adjacent GS protomers. These N-and C-termini contain the GS 269 phosphoregulatory apparatus which meet and traverse away from the GS core (Fig. 2e). When comparing human GS to previous crystal structures of yeast GS, the distance between 287 regulatory helices (a22) in adjacent monomers changes according to the activity state of GS 288 ( Fig. 3b). In the phosphorylated human GS structure, helices a22 lie 7.9 Å apart when 289 measuring Ca-Ca distances from Arg591 on chain A and Arg580 on chain B (Fig. 3b). A 290 similar measurement of the corresponding residues in the yeast proteins shows that helices 291 a22 are furthest apart, at 16 Å, when G6P is bound and GS is in its high activity state, and 292 this translates into better access for accepting the substrate 30,34 . When no G6P is bound and 293 there is no phosphorylation, GS is in the basal state and the helices lie 11 Å apart 30 . In a yeast 294 GS structure of a mimic of the inhibited state, where residues R589 and R592 were mutated 295 to Ala and GS was produced in bacteria, the helices are closest together at 8 Å 34 . This is 296 similar to the phospho-human GS, where phosphorylation appears to contribute to the closing 297 of the regulatory helices constraining the GS tetramer and thus locking it in a tense, inactive 298 state (Fig. 3b).

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The position of the extreme N-terminus is noticeably different in human and C. elegans GS 301 structures compared to yeast (Supplementary Fig. 7e). The majority of the first β-sheet in all 302 structures is in a similar orientation, however human residues before 26 (residue 7 in yeast) 303 move in the opposite direction to yeast (Supplementary Fig. 7e). This positioning of the 304 human GS N-terminus is directed towards the regulatory helices a22. Previous structural 305 investigation of C. elegans GS-GN 34 suggested a hypothesis where phosphorylation could enable the N-terminus to engage with regulatory helices, as the N-terminus is also situated 307 towards the regulatory helices 22 (Supplementary Fig. 7e). Our structure of the human, 308 phosphorylated enzyme supports this hypothesis, although the current density does not allow 309 model building before residue 13. However, using LAFTER 46 denoised maps to aid model 310 building and electron density interpretation, some density for the N-terminus is present next 311 to the regulatory helices, near to R579 and R580. This suggests that perhaps the N-terminal 312 phosphorylation sites can also interact with the regulatory helices and/or nearby residues ( Fig.   313 3a and Supplementary Fig. 7f).

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Comparisons between human, C. elegans and yeast GS structures are consistent with the 316 human structure in the inactive state. Each human GS protomer shows a closed conformation 317 of its active site, and a regulatory loop, that only becomes ordered upon G6P binding, is 318 disordered in the human structure ( Supplementary Fig. 8b and 8a). Previous studies have 319 suggested that phosphorylated tails may be able to engage the G6P binding site and directly 320 compete with G6P. However, our EM density maps show no extra density within the G6P 321 binding site (Supplementary Fig. 8a). Thus, we see no evidence to support the hypothesis 322 that the phosphorylated tails interact with residues lining the G6P pocket to directly compete Due to the flexibility evident in the N-and C-terminal tails, we were unable to build 331 phosphorylated residues in the cryo-EM map other than phospho-S641. However, we can see the beginning of the flexible phosphoregulatory "spike" region and residues from the GS "core 333 tetramer" which interact with this regulatory region (Fig. 3a, bottom right panel). To investigate 334 the relationship between allosteric regulation and inhibitory phosphorylation and elucidate the 335 mechanism of inactivation, we mutated residues in GS that contact the beginning of the 336 phosphoregulatory region. We selected residues which are not involved in G6P binding and 337 mutated these in order to "dislodge" the regulatory tails ( Fig. 3a and Supplementary Fig. 8e).

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If the phosphorylated tails are indeed holding GS in an inactivated state, weakening the 339 interaction between the core tetramer and the N-and C-termini inhibitory regions should 340 create an enzyme with higher basal activity in comparison to the WT. Consistent with our 341 hypothesis, we observed a marginal increase in basal (-G6P) GS activity in R588A+R591A, 342 Y600A, R603A, H610E and W18A mutants, that was reflective of the phosphorylated state at residues S8, S641 and S645 ( Fig. 4a and 4b). These mutants were unaffected in terms of 344 GN co-purification and with the exception of R588A+R591A mutant, they had similar melting 345 (Tm) profiles and oligomeric state to the WT GS complexes (Supplementary Fig. 3e, Fig. 4c,  Supplementary Fig. 9). All mutants except Y600A could still be activated to similar levels to 347 the WT upon addition of G6P (Fig. 4a).
348 349 Upon addition of PP1 and lambda PP, the GS mutants R588A+R591A, R603A and W18A 350 were more robustly dephosphorylated at S641 and S8 than WT GS (Fig. 4d), suggestive of 351 increased exposure of the phospho-tails to phosphatases. For the W18A mutant, this 352 dephosphorylation by both lambda-PP and PP1 resulted in over a 20-fold increase in basal 353 activity, and also an approximately 3-fold increase in comparison to WT GS (Fig. 4e). The GS 354 R603A-GN(Y195F) mutant has a basal activity similar to WT GS upon dephosphorylation.

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However, the robust dephosphorylation at S641 and S8 in GS R588A+R591A was not 356 associated with an increase in activity (Fig. 4e). As described above, R588 and R591 lie on 357 the regulatory helices and are also involved in inter-subunit interactions and form the arginine 358 cradle that interacts with phospho-S641 (Fig. 3a). In addition, we noticed some dissociation 359 of the GS R588A+R591A double mutant complex in mass photometry (Supplementary Fig.   360 9c). Therefore, the role of these residues in stabilising the GS tetramer may be the cause for 361 the lack of rescue of activity upon dephosphorylation (Fig. 4e). Moreover, dephosphorylated 362 GS had a markedly lower Tm (48 ºC) than WT or mutant GS (Fig. 4c) supporting the idea that 363 phosphorylation of the "spike" regions strengthens the inter-subunit interactions within the 364 tetramer and holding the enzyme in the "tense" conformation.  Fig. 1 and Fig. 5). The two GN dimers neighbouring a GS tetramer do not interact 376 in an identical manner, with one GS dimer tilted closer towards GS in comparison to the other 377 (Fig. 1h). This observed flexibility of GN may be aided by the variable length linkers connecting 378 the catalytic domain and the C-terminal GN 34 region that anchors GS. The precise functional 379 relevance of this movement is yet to be explored, however the linker length was shown to cradle could be strengthening the interaction between two GS protomers, thus constraining 387 the GS tetramer and leading to an inactive enzyme ( Fig. 3a and 5). Our structure also 388 suggests a role for the N-terminal phosphorylation sites S8 and S11 (sites 2 and 2a) in the 389 mechanism of human GS inactivation, as they presumably lie close to R579 and R580 on the 390 regulatory helices (Supplementary Fig. 7e and 7f). Although model building before residue 391 13 was not possible, our analyses highlight an essential role for a22 in GS inhibition, 392 specifically residues R579, R580, R588 and R591. This is consistent with previous studies showing that both N-and C-phosphorylation is required for inhibition of rabbit GS, as well as 394 there being a significant role of site 3a and site 2/2a 23, 42 . The role of the arginine cluster 395 (residues 579-591, on the a22 helices) in GS regulation was investigated in yeast orthologues, 396 revealing a role in G6P activation and suggesting a potential role in phosphorylation 397 dependent inactivation 30,34,47,48 . Our inhibited human GS structure confirms that helix a22 is 398 crucial for the phosphorylation dependent inactivation of GS, revealing that the same helix 399 a22 is involved in both allosteric activation 30 and covalent inhibition (Fig. 3 and Fig. 5). 400 401 GS phosphorylation sites lie outside of the catalytic core and within the N-and C-terminal 402 tails ( Fig. 1c and Supplementary Fig. 2). A comparison of human, C. elegans and yeast 403 structures reveals considerable differences in the position of the tails (Supplementary Fig.   404 7e). In human GS, the C-terminus is responsible for most of the observable interactions with 405 helix a22 via R588 and R591 residues, however, these residues are not conserved in C.

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elegans (Supplementary Fig. 2), perhaps explaining the positional differences between 407 human and C. elegans GS tails. Interestingly, S641 (site 3a) is also not conserved in C.  Fig. 7f and 5g). Having one tail buttressed against the regulatory 417 helix and the other steering away from the core may allow interchanging of the tails based on 418 their level of phosphorylation, perhaps explaining why multiple phosphorylation sites are 419 required. It may also aid/allow rapid dephosphorylation of GS, leading to an increase in GS 420 activity and thus promoting glycogen synthesis (Fig. 5).

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Elucidating the role of the inter-subunit domain that house the phosphorylation sites, through 423 mutations that weaken the interactions between the core tetramer and the "spike" regions, 424 resulted in basal activity equal to or higher than the WT, yet retaining activation by G6P (Fig.   425   4a). The GS Y600A mutant was not activated by G6P to the same extent as WT (Fig. 4a) yet there is little difference in the high activity (G6P-bound) state between phosphorylated and 433 dephosphorylated complexes (Fig. 2a and Fig. 4a). This is in accordance with previous 434 studies that demonstrate that G6P can overcome inhibition by phosphorylation and restore full 435 activity 2 . C-terminal mutants R588A+R591A and R603A and the N-terminal W18A mutant 436 were more easily dephosphorylated than WT (Fig. 4d) suggesting that dislodging of the 437 phosphoregulatory region leads to phosphorylation sites being more accessible to 438 phosphatases.

Fig. 5 GS and GN cooperate to synthesize glycogen
Glucose is converted into glycogen through the action of glycogenin (GN), glycogen synthase (GS) and glycogen branching enzyme (GBE). GN interacts with GS to feed the initial glucose chain into the GS active site for elongation. GS is regulated by allosteric activation and inhibitory phosphorylation. Phospho-S641 (pS641) from one C-terminal tail interacts with the regulatory helices a22 to cause enzyme inhibition. This can be relieved by G6P, with or without phosphatases, to reach a high activity state. Kinases can phosphorylate GS to inhibit the enzyme.
The robust dephosphorylation of GS R588A+R591A did not result in an increase in basal 441 activity ( Fig. 4d and 4e), which mirrors previous results in yeast 34 . The analogous mutations 442 were used in yeast GS resulting in low basal activity, yet it could still be fully activated by 443 G6P 34 . It was proposed that these residues in the regulatory helix are essential for keeping 444 GS in a "spring loaded" intermediate state, and thus charge neutralization by mutation of 445 arginine to alanine leads to the "tense" inactive state 34 . Our activity data agree with this as we 446 don't see an increase in basal activity despite dephosphorylation at S641 and S8, although 447 we do see a marginal difference between this mutant and WT in the phosphorylated state 448 ( Fig. 4a and 4e). However, it is important to note that we also see some complex dissociation 449 with the R588A+R591A mutant as evidenced by a larger dissociated complex peak in mass 450 photometry (Supplementary Fig. 9c). It is possible that the dephosphorylated R588A+R591A 451 mutant is also unstable and dissociates more easily than the phosphorylated mutant, resulting 452 in a less active preparation.

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To prevent interpretation of any artefacts created by applying D2 symmetry, the data was also 619 processed in C1 symmetry (Supplementary Fig. 6). The same particles after 2D classification 620 were subjected to 3D classification without applying symmetry. High quality classes containing 783,177 particles were un-binned to a box size of 288 pixels and then subjected to 3D 622 refinement and postprocessing, to generate a 3.1 Å map. Following iterative rounds of contrast 623 transfer function refinement and Bayesian particle polishing to generate a 2.8 Å map.

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To elucidate the movement of phosphoregulatory regions, an alignment free 3D classification 626 with a mask containing the "spike" density was performed, using a regularisation parameter T 627 of 60 59 (Supplementary Fig. 5f and 5g).  Fig. 6e and 6e).