Key Role of the Carboxyl Terminus of Hyaluronan Synthase in

Subscriber access provided by UB + Fachbibliothek Chemie | (FU-Bibliothekssystem)

Article

Key role of the carboxyl terminus of hyaluronan synthase in processive synthesis and size control of hyaluronic acid polymers Ji Yang, FANGYU CHENG, Huimin Yu, Junting Wang, Guo zhi gang, and Gregory Stephanopoulos Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b01239 • Publication Date (Web): 13 Feb 2017 Downloaded from http://pubs.acs.org on February 14, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Biomacromolecules is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

3

Key role of the carboxyl terminus of hyaluronan synthase in processive synthesis and size control of hyaluronic acid polymers

4

Ji Yang# , Fangyu Cheng#1, Huimin Yu*1, , Junting Wang1, Zhigang Guo1,

5

and Gregory Stephanopoulos*3

6

1 Key Laboratory for Industrial Biocatalysis of the Ministry of Education, Department

7

of Chemical Engineering, Tsinghua University, Beijing 100084, P. R. China

8

2 Center for Synthetic and Systems Biology, Tsinghua University, Beijing 100084, P.

9

R. China

10

3 Department of Chemical Engineering, Massachusetts Institute of Technology,

11

Cambridge, MA 02139, USA

1 2

1

2

12

#

These two authors contributed equally to this work. To whom correspondence should be addressed. E-mail: [email protected] (H.Y.); [email protected] (G.S.). Author contributions: H.Y. and G. S. designed the research; J.Y. performed the computational research and data analysis; F.C., J.W. and Z.G. performed the experimental research; and all authors wrote or revised the paper. The authors declare no competing financial interest. *

1

ACS Paragon Plus Environment

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 39

13

Abstract

14

The essential pathophysiological roles of hyaluronic acid (HA) strongly depend on

15

HA binding and HA size. Here we deployed the atomic vision of molecular dynamics

16

(MD) simulation to experimentally investigate the influence of C-terminal mutations

17

of Streptococcus equisimilis hyaluronan synthase (SeHAS) on HA product synthesis

18

in Escherichia coli. R413 was vital for HA production, as the removal or mutation of

19

R413 led to inactivation of SeHAS. MD simulations indicated that R406-R413

20

constituted an HA-binding pattern that stabilized the HA-SeHAS complex. We further

21

increased HA product size via site-directed mutation of the SeHAS C-terminal

22

residues 414 to 417 based on the hypothesis that higher binding affinity between the

23

SeHAS C-terminus and HA would lead to larger HA size, underlying the important

24

role of the HA-SeHAS interaction in HA size control. W410A and T412A mutations

25

also

26

catalysis-transformation-translocation model was proposed for the HA synthesis and

27

translocation processes.

28

Keywords: HA synthesis, molecular dynamics simulation, C-terminal mutation,

29

HA-SeHAS interaction, HA size control model

completely

deactivated

SeHAS.

Moreover,

a

30 31

INTRODUCTION

32

Class I hyaluronan synthases (HASs), which employ processive mechanisms to

33

polymerize hyaluronic acid (HA) chains, are remarkable, membrane-embedded 2


Page 3 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

34

β-glycosyltransferases, that catalyze the biosynthesis of HA from two distinct

35

substrates while also translocating HA chains across the cell membrane within the

36

same proteins 1. Multiple HAS proteins have been identified in bacteria, mammals,

37

amphibians, avians and even viruses. HA, a linear and negatively charged

38

glycosaminoglycan (GAG), is ubiquitously expressed in the mammalian extracellular

39

matrix (ECM) and is utilized for a wide range of a wide range of cosmetic, health, and

40

clinical applications. It is composed of repeating disaccharide units containing

41

D-glucuronic

42

mass ranging from 500 kDa to 10,000 kDa. The high hydration levels of HA confer

43

suitable properties upon the ECM to facilitate motility and proliferation of cells. In

44

addition to its structural functions, which include the stabilization of cell-free spaces,

45

fluid retention and tissue hydration, HA also participates in cell signaling via

46

interactions with membrane receptors such as CD44 and Toll-like receptors (TLRs),

47

modulating many important processes including morphogenesis, inflammation,

48

tumorigenesis, migration and apoptosis 2, 3.

acid (GlcUA) and N-acetyl-D-glucosamine (GlcNAc), with a molecular

49

HA chain size is critical under many physiological and pathophysiological

50

conditions 4, 5. Different sizes of HA can induce different signaling pathways 6-8. It has

51

been proposed that large HA molecules (>1000 kDa) facilitate the clustering of

52

receptors in the cell membrane to exert anti-apoptotic and anti-angiogenic activities 7,

53

9

54

HA molecules ( K414 > K415, site-directed mutagenesis was

302

carried out (Figure 4 and Table S1). As expected, the HA titers of R406A and 16


Page 16 of 39

Page 17 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

303

R413A variants were greatly decreased. Surprisingly, when the arginine was changed

304

to a lysine, the R413K variant barely produced HA as well. Based on an initial

305

α-helical structure (Figure S9), MD simulation of the R413K C-terminal peptide

306

interacting with HA indicated that the important role of residue 413 in HA-binding

307

was drastically reduced when it was changed to a lysine (Figure S10). The

308

trajectories of the simulation also revealed that the R413K C-terminal peptide bound

309

loosely to HA (it was found to detach several times over the course of an 80-ns

310

simulation).

311 312

Figure 4. Titers (A) and size distribution (B) of HA products produced by SeHAS

313

variants constructed via site-directed mutagenesis. The arrow in (B) indicates the HA

314

Mw of WT SeHAS and different SeHAS variants, with the exception of R413A and

315

R413K. All experiments were performed three times.

316

The mutations K414A, K414R, K415A, and K415R did not notably affect the HA

317

titers of these variants significantly, suggesting that K414 and K415 are not vital for 17


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

318

HA production by SeHAS. However, we also noted that the Mw of the HA produced

319

by the K414R variant was significantly increased compared to the original Mw, rising

320

from 790 kDa to 1270 kDa. According to the simulation with the WT C-terminal

321

peptide, K414 may also be involved in the interaction with HA (Figure 2D). We

322

hypothesized that the mutation of K414 to an arginine enhanced the interaction

323

between the SeHAS C-terminus and HA and subsequently enlarged the HA product

324

size.

325

Rational design to increase the HA product size

326

To further explore the relationship between the SeHAS C-terminal structure,

327

HA-binding affinity, and SeHAS function, we sought to increase the HA product size

328

by rational design based on our assumption that higher binding affinity between the

329

SeHAS C-terminus and HA would lead to larger HA products. To increase the

330

SeHAS-HA binding affinity, L416 and L417 were selected as two other potential

331

HA-binding sites. On the one hand, they are in close proximity to the charged

332

R406-R413 HA-binding pattern in the helical wheel (Figure 3B). On the other hand,

333

they sequentially fall outside of the range of R406 to R413 and may extend the

334

original HA-binding region. To investigate the effects of net charge variations, we

335

also added between one and four arginine(s) after R413.

336

The differences in HA-binding free energy between the WT C-terminal peptide of

337

SeHAS and several point-mutation variants (R413K, K414R, K415R, L416R, and

338

L417R) were calculated by performing free energy perturbation (FEP) simulations

339

(Figure S11 and Table S2). Changes in the HA-binding affinities of R413K, K414R, 18


Page 18 of 39

Page 19 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

340

and K415R were consistent with the experimental results (Table S1), although these

341

changing values might be underestimated (R413K) or overestimated (K415R) given

342

that the simulated system is different from the real system. The primary difference

343

between the simulated and real systems is the binding restraint: in simulation, the HA

344

molecule was able to freely bind the C-terminal peptide, adopting various

345

conformations, while in reality, the SeHAS scaffold would restrain its accessibility to

346

potential HA-binding sites and the SeHAS C-terminus would also interact with other

347

parts of SeHAS in addition to HA. FEP calculations with the L416R and L417R

348

variants predicted that these two mutations might enhance HA-binding affinities

349

(Table S2).

350

The coordination number (CN) of each basic residue in the SeHAS C-terminal

351

peptide was also calculated to assess the details of interactions with HA (Table 1).

352

CN represents the number of carboxyl oxygens in HA that coordinate with the overall

353

side chain for each basic residue in the C-terminal peptide, according to the following

354

equation:

355

CN =

∑c

ij

( r −r i

j∈G2

j min

) , ri − rj min = min { ri − rj } , cij i∈G 1

( ( r −r )= 1− ( r − r

) d )

1 − ri − rj d 0

i

m

j

i

n

j

0

356

where G1 are nitrogens in the basic side chain of the SeHAS C-terminal peptide, G2

357

are carboxyl oxygens in HA, |ri - rj| is the distance between i and j atoms, and other

358

parameters are default values (d0 = 4.0 Å, n = 6, and m = 12), similar to the CN

359

defined by NAMD

360

the following order, which is indicative of their importance for HA-binding: 406 ≈

37, 45

. In general, the CNs of the C-terminal residue sites assumed

19


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 39

361

413 > 416 > 414 ≈ 417 > 415 (Table 1). It is indicated that L416 would likely interact

362

with HA closely if it was mutated to an arginine or lysine and consequently increased

363

the HA product size. CNs of the basic residues in the SeHAS C-terminal peptide

364

surrounding both the carboxyl and hydroxyl oxygens in HA suggested similar results

365

(Table S3).

366

Table 1. CNs between carboxyl oxygens in HA and the amine group nitrogens of the

367

basic residues in the SeHAS C-terminal peptide calculated from MD simulations. a

SeHAS variant

d

406 R 3.13±1.20

413

414

415

R 2.16±0.72

K 0.85±1.26

K 0.01±0.06

R 0.67±0.59

K 0.02±0.06

K 0.33±0.67

K 0.46±0.63

R 1.64±0.60

R 1.75±0.27

R 0.29±0.52

K 0.01±0.10

K415R

R 3.00±0.76

R 0.88±0.77

K 0.01±0.03

R 0.00±0.00

L416K

R 1.99±0.68

R 1.99±0.63

K 0.01±0.01

K 0.06±0.27

K 0.42±0.65

L416R

R 2.01±0.83

R 1.58±0.51

K 0.01±0.02

K 0.50±0.66

R 2.18±1.25

L417K

R 1.83±0.94

R 0.43±0.73

K 0.23±0.49

K 0.00±0.02

K 0.23±0.35

L417R

R 0.87±0.90

R 0.99±0.72

K 0.48±0.94

K 0.04±0.24

R 2.86±0.86

R413RKKLL

R 2.69±0.89

R 1.59±0.54

R 0.01±0.03

K 0.00±0.00

K 0.73±0.74

R413RRKKLL

R 1.60±0.69

R 2.90±0.65

R 1.89±1.01

R 0.01±0.02

K 0.86±0.77

K 0.06±0.25

R413RRRKKLL

R 1.61±0.55

R 1.62±0.53

R 0.02±0.05

R 1.49±1.00

R 2.11±0.75

K 0.20±0.51

K 0.04±0.11

R413RRRRKKLL

R 1.97±0.85

R 2.21±0.46

R 0.02±0.03

R 0.67±0.86

R 2.66±0.68

R 0.42±0.50

K 0.01±0.02

WT

b

R413K K414R

368 369

c

e

416

417

a

Values shown are the means±S.E.

b

CNs of WT SeHAS and R413 were calculated from 80-ns ABF simulations. 20


418

419

K 0.36±0.57

Page 21 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

370 371 372 373

c

CNs of other variants were calculated from unbiased 40-ns MD simulations.

d

Residues that interacted strongly (CN > 1.0) and moderately (CN = 0.5~1.0) with HA are

highlighted in black and grey, respectively. e

Mutant residues are underlined.

374

Site-directed mutagenesis revealed that the Mw of HAs produced by L416K,

375

L416R, and L417K variants increased to ~1800 kDa as expected, although the HA

376

Mw of L417R remained at 750 kDa (Figure 5 and Table S4). The unchanged HA size

377

of L417R might be attributable to the disturbance in the original binding pattern (the

378

CNs of both R406 and R413 decreased in L417R), although the interaction between

379

R417 and HA was strongly enhanced (Table 1). HA titers of all four variants

380

decreased to almost half of the WT. When the two single mutations K414R and

381

L416K were combined, R413RKKLL produced even larger HA with a Mw of 2290

382

kDa, suggesting that there was a collective effect of R414 and K416 on HA-binding

383

affinity. However, when the positive charge of the SeHAS C-terminus was further

384

increased via the insertion of two to four arginine residues after R413, the HA Mw of

385

R413RRKKLL, R413RRRKKLL, and R413RRRRKKLL remained at ~2300 kDa; thus,

386

net charge did not play a predominant role in determining HA-binding affinity and

387

HA product size.

21


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

388 389

Figure 5. Titers (A) and size distribution (B) of the HA products produced by SeHAS

390

variants constructed via site-directed and inserted mutagenesis. The arrows in (B)

391

indicates the HA Mw of WT SeHAS and different SeHAS variants. All experiments

392

were performed three times.

393

Extension of the SeHAS C-terminus with one alanine (neutral) or glutamic acid

394

(acidic) after R413 (R413AKKLL and R413DKKLL) were also performed. Results

395

showed that the variants produced HA of similar size around 1900 kDa, which further

396

confirming the importance of L416K mutation (Figure S11).

397

Additionally, to eliminate the effect that may be caused by different protein

398

expression levels, six representative SeHAS variants in this study (R413RRRRKKLL,

399

WGT412, WGTR413, R406A, R413K and L416R), along with WT SeHAS (positive

400

control) and original plasmid pMBAD (negative control), were analyzed by western

401

blot (Figure S13). All variants, even the ones that hardly produce HA (WGT412 and

402

R413K), were expressed successfully. Moreover, the OD600 of all the mutants were

22


Page 22 of 39

Page 23 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

403

similar with 8 h and 24 h fermentation, indicating a similar expression level of all the

404

SeHAS variants in this work.

405 406

DISCUSSION

407

Comparison between arginine and lysine for HA-binding

408

When a residue was mutated from lysine to arginine (e.g., K414R), this generally

409

enhanced the HA-binding affinity, and vice versa (e.g., R413K). Compared with

410

lysine, arginine is more favorable for HA-binding. First, the guanidine group in

411

arginine can form a bidentate hydrogen bond with the carboxyl group in HA, while

412

the amine group in lysine can only form a monodentate hydrogen bond (Figure S7).

413

Second, given that arginine contains five hydrogen bond donors (guanidine

414

hydrogens) while lysine only contains three (amine hydrogens), the potential

415

interaction space of arginine is larger than that of lysine. Third, although the side

416

chain of arginine is less flexible than that of lysine, the side chain of arginine is longer

417

(six-bond length in arginine and five-bond length in lysine).

418

Lysine and arginine assume important roles in HA translocation and HA synthesis

419

reaction, which are two critical functions of SeHAS, respectively. The standard

420

deviations of the CNs of lysine calculated via MD simulations are generally

421

comparable to or even larger than their mean values (Table 1 and Figure S14); thus,

422

compared with arginine, lysine residues have the potential to more easily bind to and

423

unbind from the HA chain. This transient HA interaction is exactly what is required

424

for the HA translocation process: the HA chain must attach to and detach from

23


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

425

SeHAS to enable rapid on/off rates and progressive translocation to the cell exterior.

426

The smaller CN deviations of arginine compared to their mean values suggest that

427

arginine residues can stabilize the conformation of the HA-SeHAS complex upon

428

interaction with the HA chain and thus become important binding sites during the HA

429

synthesis reaction, particularly if they form HA-binding patterns such as R406-R413.

430

Roles of R406 and R413 in SeHAS C-terminus

431

To our knowledge, it is the first time that the function of the SeHAS C-terminus

432

have been investigated. When R413 was removed (Figure 1) or mutated (Figure 4),

433

HA titers could hardly be detected, including the variants K413RKLL and

434

K413RRRRKKLL (data not shown), emphasizing the critical role of R413 in HA

435

synthesis. The HA titer of R406A was heavily decreased as well, while its HA Mw

436

was maintained at 790 kDa. R406 and R413 could form a binding doublet to enable

437

strong binding with HA (Figure 3); thus, these residues contribute a high binding

438

force to hold the HA chain firmly in the binding sites of SeHAS and likely play a

439

significant role in the catalysis necessary to transfer one type of substrate to the HA

440

chain. Disturbance of the crucial R406-R413 (RX6R) binding pattern renders catalysis

441

less efficient (R406A) or inactivates it (WGT412, R413A, and R413K).

442

Notably, changing the arginine to a lysine at residue 413 severely disrupted the

443

original R406-R413 binding pattern (Table 1) and the HA-binding affinity of R413K

444

decreased (Table S2). Moreover, the R406-R413 motif contributed to the stabilization

445

of the local α-helical structure. When bound to HA in simulations, the α-helical

24


Page 24 of 39

Page 25 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

446

content of WT SeHAS greatly increased from 61.4% to 81.2%, while that of R413K

447

only increased by 4.3% (from 64.6% to 68.9%) (Figure S15).

448

Sequence alignment of HASs showed that the R/K406 and R413 were conserved

449

across bacterial and almost all species, respectively (Figure S16). Moreover, we

450

noticed that the “410-WGT-412” motif was also highly conserved. Site-directed

451

mutagenesis showed that the change of W410 and T412 to alanine (W410A and

452

T412A) completely deactivated SeHAS and no HA products could be detected. As a

453

speculation, Trp and Thr residues in the WGT motif may contribute CH/π and/or

454

classic hydrogen bond with HA, thereby co-assisting the specific bidentate hydrogen

455

bond interaction between R413-HA, or maintain the stability of SeHAS scaffold.

456

More precise inspection will be perform on the “WGT” and other conserved regions

457

in SeHAS in the future.

458

To verify the hypothesis that RX6R can form an HA-binding pattern, binding

459

affinity tests of RX6R-containing peptides toward HA were performed with four

460

peptides in similar length and labeled by FITC at the N-terminus (Table S5). Besides

461

the C-terminal peptide of SeHAS (KL-20, 398-KX7RX6RKKX2-417 in SeHAS),

462

another RX6R-containing peptide (YK-22, 292-X2KX3RX6RX6KK-313 in SeHAS)

463

that contains a highly conserved RX6R pattern among the Class I HASs (Figure S16)

464

was also selected. DK-20 (269-X3KX7KX7K-288 in SeHAS) and AY-19

465

(AAQSVTGNILVCSGPLSVY) were control peptides with and without basic

466

residues, respectively.

25


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

467

A strong chelating binding was detected in both KL-20/HA and YK-22/HA

468

complexes after mixing the solution of KL-20 and YK-22 with HA (Figure S17A).

469

The binding strength of the four peptides with HA was further assessed through the

470

holding capability test using HA-polyacrylamide (PAM) gel. For KL-20 or YK-22,

471

the interaction between the RX6R-containing peptide and HA was so strong that

472

precipitants were formed even in the HA-PAM gel (Figure S18). Quantification

473

analyses showed that the molar holding ratios of HA to YK22, KL20 and DK20 in the

474

gel were around 1:34, 1:26 and 1:5, respectively, confirming that the strong chelating

475

interaction only occurred between the guanidine groups of arginine in RX6R motif

476

and the carboxyl groups of HA as shown in Figure 2A. For control peptides DK-20

477

and AY-19, stronger fluorescence was observed in the DK-20/HA-PAM gel than that

478

of AY-19/HA-PAM (Figure S17C and Figure S18), indicating that basic residues

479

can also play a role in HA binding, although the binding affinity is greatly lower than

480

RX6R-HA binding affinity.

481

Roles of the residues 414-417 in SeHAS C-terminus

482

The HA Mw finally increased about three-fold, from 790 kDa (in WT SeHAS) to

483

2290 kDa, by rational mutation(s) in residue 414 to 417, indicating that larger HA

484

products could be obtained by rationally enhancing the HA-binding affinity of the

485

SeHAS C-terminus (Figure 5), likely favoring HA chain retention, slowing down the

486

HA translocation process and elongating the time required for HA synthesis. We also

487

observed that HA Mw and titers of SeHAS variants involving mutation(s) in residues

488

414 to 417 were highly negatively correlated (Figure S19), implying that HA size 26


Page 26 of 39

Page 27 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

489

might be affected by HA synthesis efficiency, which in turn was based on HA

490

translocation efficiency. The two exceptions, WGTR413 and L417R, might be

491

attributable to the disturbance of the original R406-R413 binding pattern, which is

492

important for catalytic efficiency. Additionally, interactions between the C-terminus

493

and other parts of SeHAS should also be taken into consideration.

494

The mechanism underlying HA size control remains unclear because many intricate

495

factors can affect HA product size, including alterations in the HAS structure and

496

micro-environmental conditions. The HAS structure can be altered by mutagenesis 17,

497

18

498

glycosylation) 2, 46, or binding to regulator molecules 47. The environmental conditions

499

surrounding HAS constitute another important factor that influences HA product size.

500

Presumably, substrate availability (i.e., how rapidly HAS can recruit sugar-UDP

501

substrates to its reaction center) controls HA size based on the evidence that smaller

502

HAs will be generated if the concentration ratio of HA to HAS decreases

503

the micro-environmental viscosity increases due to the enhancement of glycerol

504

content

505

vivo differs significantly from its behavior in vitro. In our experiments with intact E.

506

coli cells, the Mw of HA produced in vivo was less than 2500 kDa, while the in vitro

507

HA Mw could be as large as ~3100 kDa when isolated membranes were employed

508

(Figure S5B) and all the variants demonstrated similar HA Mw (data not shown).

509

These results are likely attributable to different levels of substrate availability due to

510

the viscous cytoplasmic environment and, potentially, low substrate concentration in

,

post-translational

modifications

(e.g.,

18

, or if a UDP inhibitor is present

phosphorylation,

ubiquitination

or

13, 14, 16

, if

16

. Moreover, the behavior of SeHAS in

27


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 39

511

vivo. Other possible reasons may include the limited space in the periplasm of intact

512

E. coli cells and curvature change of the cell membranes. We also observed a negative

513

correlation between HA synthesis efficiency and HA size in vivo (Figure S19), but a

514

previous in vitro study provided a contradictory conclusion: HAS polymerization

515

activity and HA product size can be independently manipulated

516

differences, there are likely multiple mechanisms to control HA product size, and

517

different restrictions may take effect either in vivo or in vitro.

518

Proposed mechanism for HA production and HA size variation

18

. Given these

519

Various models depicting HAS architecture and mechanisms of HA synthesis have

520

been discussed, but the actual details remain unknown due to the lack of information

521

regarding the three-dimensional structures of HAS. Here, we propose a

522

catalysis-transformation-translocation model (Figure 6), specifically highlighting the

523

function of the SeHAS C-terminal region, to explain how SeHASs synthesize polymer

524

chains and control product size. This model can also expand our understanding to the

525

synthesis mechanism of other processive β-glycosyl polymerases, such as cellulose

526

and chitin synthases.

28


Page 29 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

527 528

Figure 6. The schematic catalysis-transformation-translocation model proposed for

529

HA synthesis and the control of HA size by SeHAS. (A) When the substrate

530

(sugar-UDP) binds in the active sites, the glycosyl transfer reaction is initiated. (B)

531

Completion of the reaction after time tcat triggers the transformation of SeHAS into a

532

translocation conformation, the release of the UDP reaction product, and the

533

translocation of HA to the cell exterior. (C) Binding of another sugar-UDP substrate

534

in the active sites induces SeHAS to assume a catalysis conformation such that the

535

HA chain is restricted to translocate only at a distance of one sugar to bind firmly with

536

SeHAS

537

catalysis-transformation-translocation cycle begins. The catalytic and pore regions of

and

initiate

another

reaction.

29


(D)

Another

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

538

SeHAS are shown as a pink oval and a pink box, respectively. The SeHAS C-terminal

539

peptide is shown as a red helix. The HA chain containing alternating GlcNAc (blue

540

diamonds) and GlcUA (green circles) interacts with both regions of SeHAS, including

541

its C-terminus. The turbulent exterior force Frelease and the binding and retaining

542

forces from SeHAS are indicated with arrows. Local conformational variations in

543

SeHAS (red shape) alter HA binding affinity.

544

Each synthesis cycle of adding one sugar (disregarding the difference between

545

GlcNAc and GlcUA) to the HA chain consists of two steps. First, when the substrate

546

(GlcNAc-UDP or GlcUA-UDP) binds in the active sites, the glycosyl transfer reaction

547

is performed in time tcat. During this step, the exterior forces Frelease, which may be

548

derived from Brownian motion, shear force generated via cell movement, or

549

interactions with an external matrix, attempt to pull the HA chain away from SeHAS

550

18, 26

551

chain firmly bound to SeHAS. Here, the C-terminal R406-R413 binding pattern

552

greatly contributes to Fbinding. Successful completion of the sugar transfer reaction

553

requires Frelease ≤ Fbinding during this step.

, while the Fbinding interactions between HA and SeHAS attempt to keep the HA

554

Second, completion of the reaction in the first step triggers the transformation of

555

SeHAS to a “translocation conformation”, which decreases its binding affinity to the

556

HA chain. Subsequently, the UDP product departs from the binding sites and the HA

557

chain begins to move to the exterior, although it is not known if HA translocation is

558

associated with UDP loss. When another sugar-UDP substrate binds in the active

559

sites, SeHAS is further adjusted to a “catalysis conformation”, which restricts the HA 30


Page 30 of 39

Page 31 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

560

chain to translocate at a distance of only one sugar to enable firm binding with

561

SeHAS. The time required for HA translocation at one sugar distance, ttrans, is affected

562

by both Frelease and the retention force Fretain (Figure 6B). The time required for the

563

sugar-UDP substrate to diffuse to and bind with the active sites of SeHAS is tdiff. The

564

total time required for one cycle is tcycle = tcat + ttrans, representing the efficiency of HA

565

synthesis and the apparent activity of SeHAS. If the HA chain translocates more than

566

one sugar distance before another substrate binds SeHAS, it will continue to move

567

and

568

catalysis-transformation-translocation cycle, the sugar-UDP substrate should diffuse

569

to and bind with SeHAS within a duration of time where tdiff ≤ tcycle.

consequently

leave

the

SeHAS.

Thus,

to

begin

another

570

In our study, HA size increased upon mutagenesis of the SeHAS C-terminus

571

(residue 414 to 417), resulting in the enhancement of Fretain and thus increased tcycle. In

572

previous studies performed by the Weigel group, HA size decreased as the viscosity

573

of its micro-environment increased

574

decreased 16, which in turn reduced local substrate availability and increased the time

575

tdiff. The different sizes of HA produced in vivo (Figure 1B) and in vitro (Figure

576

S5B) may be attributable to their differential substrate availabilities, leading to

577

different tdiff. As HA synthesis efficiency is highly correlated with HA size in vivo

578

(Figure S19), whereas a contrasting conclusion was drawn in vitro 18, we speculated

579

that in vivo HA size was predominantly controlled by the restriction tdiff ≤ tcycle. When

580

HA Mw increased to ~3500 kDa (in vitro), both Frelease ≤ Fbinding and tdiff ≤ tcycle could

581

take effect to limit HA size.

18

or as the concentration ratio of HA to HAS

31


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

582

Page 32 of 39

CONCLUSIONS

583

In conclusion, the residue sites investigated in this study are classified into two

584

categories according to their functions: R406 and R413 are primarily involved in

585

catalysis, while the other residues between 414 and 417 are involved in HA

586

translocation. In our catalysis-transformation-translocation model (Figure 6), the

587

residues involved in the catalysis conformation are not necessarily involved in

588

defining the translocation conformation, although one residue might contribute to

589

both processes. HAS activity requires a shorter tcycle to be efficient, while larger HA

590

production requires a longer tcycle to overcome the restriction tdiff ≤ tcycle in vivo.

591

Disturbance of the R406-R413 binding pattern renders catalysis less efficient

592

(R406A) or inactivates it (WGT412, R413A, and R413K), whereas restrictions on

593

another substrate (t2,diff ≤ t2,cycle) would allow HA size to remain unchanged. Residues

594

414 to 417 are generally involved in HA translocation to the cell exterior. Increased

595

HA-binding affinity leads to a longer ttrans and in turn tcycle; thus, it takes more time to

596

complete one catalysis-transformation-translocation cycle and produce larger HA

597

chains. The mechanism described in this study will also inform our understanding of

598

the polymer size control mechanism for other processive glycosyltransferases, such as

599

cellulose synthase and chitin synthase.

600 601

ASSOCIATED CONTENT

602

Supporting Information.

603 604

RX6R-containing peptides and binding affinity evaluation to HA by HA-PAM gel, mathematical

catalysis-transformation-translocation

model

32


proposed

for

HA

Page 33 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

605

production. This material is available free of charge via the Internet at

606

http://pubs.acs.org.

607 608

AUTHOR INFORMATION

609

Corresponding Author

610

*

611

(H.Y.); [email protected] (G.S.).

612

Author Contributions

613

#

614

H.Y. and G. S. designed the research; J.Y. performed the computational research and

615

data analysis; F.C., J.W. and Z.G. performed the experimental research; and all authors

616

wrote or revised the paper.

617

The authors declare no competing financial interest.

To whom correspondence should be addressed. E-mail: [email protected]

These authors contributed equally.

618 619

ACKNOWLEDGMENTS

620

This work was supported by the National Key Basic Research Project 973

621

(2013CB733600), the National Natural Science Foundation of China (No. 21476126;

622

No. 20976094), and the China Postdoctoral Science Foundation (No. 2015M581110).

623 624 625

REFERENCES 1. Weigel, P. H.; DeAngelis, P. L. J Biol Chem 2007, 282, 36777-36781.

33


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

626 627

628 629

2. Vigetti, D.; Karousou, E.; Viola, M.; Deleonibus, S.; De Luca, G.; Passi, A. Bba-Gen Subjects 2014, 1840, 2452-2459.

3. Suzuki, T.; Suzuki, M.; Ogino, S.; Umemoto, R.; Nishida, N.; Shimada, I. P Natl Acad Sci USA 2015, 112, 6991-6996.

630

4. Jiang, D.; Liang, J.; Noble, P. W. Annu Rev Cell Dev Bi 2007, 23, 435-461.

631

5. Jiang, D. H.; Liang, J. R.; Noble, P. W. Physiol Rev 2011, 91, 221-264.

632

6. Itano, N. J Biochem-Tokyo 2008, 144, 131-137.

633

7. Jiang, D. H.; Liang, J. R.; Fan, J.; Yu, S.; Chen, S. P.; Luo, Y.; Prestwich, G. D.;

634

Mascarenhas, M. M.; Garg, H. G.; Quinn, D. A.; Homer, R. J.; Goldstein, D. R.;

635

Bucala, R.; Lee, P. J.; Medzhitov, R.; Noble, P. W. Nat Med 2005, 11, 1173-1179.

636

8. Stern, R.; Asari, A. A.; Sugahara, K. N. Eur J Cell Biol 2006, 85, 699-715.

637

9. Deed, R.; Rooney, P.; Kumar, P.; Norton, J. D.; Smith, J.; Freemont, A. J.;

638

Kumar, S. Int J Cancer 1997, 71, 251-256.

639

10.

Medina, A. P.; Lin, J. L.; Weigel, P. H. Bmc Biochem 2012, 13.

640

11.

Rooney, P.; Wang, M.; Kumar, P.; Kumar, S. J Cell Sci 1993, 105, 213-218.

641

12.

Csoka, A. B.; Frost, G. I.; Stern, R. Matrix Biol 2001, 20, 499-508.

642

13.

DeAngelis, P. L. Curr Pharm Biotechno 2008, 9, 246-248.

643

14.

Jing, W.; DeAngelis, P. L. J Biol Chem 2004, 279, 42345-42349. 34


Page 34 of 39

Page 35 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

644 645

15.

43, 9234-9242.

646

16.

647

243-51.

648

17.

649

Tlapak-Simmons, V. L.; Baron, C. A.; Weigel, P. H. Biochemistry-Us 2004,

Baggenstoss, B. A.; Weigel, P. H. Analytical biochemistry 2006, 352,

Kumari, K.; Baggenstoss, B. A.; Parker, A. L.; Weigel, P. H. J Biol Chem

2006, 281, 11755-11760.

650

18.

Weigel, P. H.; Baggenstoss, B. A. Glycobiology 2012, 22, 1302-1310.

651

19.

Heldermon, C.; DeAngelis, P. L.; Weigel, P. H. J Biol Chem 2001, 276,

652

2037-2046.

653

20.

Hofman, K.; Stoffel, W. Biol Chem Hoppe-Seyler 1993, 374, 166.

654

21.

Tlapak-Simmons, V. L.; Kempner, E. S.; Baggenstoss, B. A.; Weigel, P. H. J

655

656 657

658 659

Biol Chem 1998, 273, 26100-26109.

22.

Hubbard, C.; McNamara, J. T.; Azumaya, C.; Patel, M. S.; Zimmer, J. J Mol

Biol 2012, 418, 21-31.

23.

Tlapak-Simmons, V. L.; Baron, C. A.; Gotschall, R.; Haque, D.; Canfield,

W. M.; Weigel, P. H. J Biol Chem 2005, 280, 13012-13018.

660

24.

Prehm, P. Biochem J 2006, 398, 469-473.

661

25.

Breton, C.; Imberty, A. Curr Opin Struc Biol 1999, 9, 563-571.

662

26.

Weigel, P. H. International Journal of Cell Biology 2015, Article ID 367579. 35


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

663 664

665

27.

Lairson, L. L.; Henrissat, B.; Davies, G. J.; Withers, S. G. Annu Rev Biochem

2008, 77, 521-555.

28.

Sambrook, J.; Russell, D., Molecular Cloning, A Laboratory Manual, the

666

third edition, Cold Spring Horbor laboratory Press. In Cold Spring Harbor, New

667

York: 2001.

668 669

29.

Alberti, S.; Ashbaugh, C. D.; Wessels, M. R. Mol Microbiol 1998, 28,

343-353.

670

30.

Yu, H. M.; Stephanopoulos, G. Metab Eng 2008, 10, 24-32.

671

31.

Diferrante, N. J Biol Chem 1956, 220, 303-306.

672

32.

Chong, B. F.; Nielsen, L. K. J Biotechnol 2003, 100, 33-41.

673

33.

Cheng, F. Y.; Gong, Q. Y.; Yu, H. M.; Stephanopoulos, G. Biotechnol J

674

675 676

677 678

679

2016, 11, 574-584.

34.

Bodevin-Authelet, S.; Kusche-Gullberg, M.; Pummill, P. E.; DeAngelis, P.

L.; Lindahl, U. J Biol Chem 2005, 280, 8813-8818.

35.

Jorgensen, W. L.; Chandrasekhar, J.; Madura, J. D.; Impey, R. W.; Klein, M.

L. J Chem Phys 1983, 79, 926-935.

36.

Brooks, B. R.; Brooks, C. L.; Mackerell, A. D.; Nilsson, L.; Petrella, R. J.;

680

Roux, B.; Won, Y.; Archontis, G.; Bartels, C.; Boresch, S.; Caflisch, A.; Caves, L.;

681

Cui, Q.; Dinner, A. R.; Feig, M.; Fischer, S.; Gao, J.; Hodoscek, M.; Im, W.; Kuczera, 36


Page 36 of 39

Page 37 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

682

K.; Lazaridis, T.; Ma, J.; Ovchinnikov, V.; Paci, E.; Pastor, R. W.; Post, C. B.; Pu, J.

683

Z.; Schaefer, M.; Tidor, B.; Venable, R. M.; Woodcock, H. L.; Wu, X.; Yang, W.;

684

York, D. M.; Karplus, M. J Comput Chem 2009, 30, 1545-1614.

685 686

687 688

37.

Phillips, J. C.; Braun, R.; Wang, W.; Gumbart, J.; Tajkhorshid, E.; Villa, E.;

Chipot, C.; Skeel, R. D.; Kale, L.; Schulten, K. J Comput Chem 2005, 26, 1781-1802.

38.

Feller, S. E.; Zhang, Y. H.; Pastor, R. W.; Brooks, B. R. J Chem Phys 1995,

103, 4613-4621.

689

39.

Darden, T.; York, D.; Pedersen, L. J Chem Phys 1993, 98, 10089-10092.

690

40.

Tuckerman, M.; Berne, B. J.; Martyna, G. J. J Chem Phys 1992, 97,

691

1990-2001.

692

41.

Bennett, C. H. J Comput Phys 1976, 22, 245-268.

693

42.

Humphrey, W.; Dalke, A.; Schulten, K. J Mol Graph Model 1996, 14, 33-38.

694

43.

Luscombe, N. M.; Laskowski, R. A.; Thornton, J. M. Nucleic Acids Res

695

696 697

2001, 29, 2860-2874.

44.

Mishra, A.; Gordon, V. D.; Yang, L. H.; Coridan, R.; Wong, G. C. L. Angew

Chem Int Edit 2008, 47, 2986-2989.

698

45.

Iannuzzi, M.; Laio, A.; Parrinello, M. Phys Rev Lett 2003, 90.

699

46.

Vigetti, D.; Viola, M.; Karousou, E.; Deleonibus, S.; Karamanou, K.; De

700

Luca, G.; Passi, A. Febs J 2014, 281, 4980-4992. 37


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

701 702

47.

Bourguignon, L. Y. W.; Gilad, E.; Peyrollier, K. J Biol Chem 2007, 282,

19426-19441.

38


Page 38 of 39

Page 39 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

Biomacromolecules

 Table of Contents graphic

2

3 4 5 6 7 8 9 10

Mutagenesis experiments and molecular dynamics simulations revel that the Cterminus of Streptococcus equisimilis hyaluronan synthase comprises a novel HAbinding pattern and plays an important role in the processive synthesis and size control of hyaluronic acid polymers.


Key Role of the Carboxyl Terminus of Hyaluronan Synthase in

Recommend Documents