Structural Insight into the Stabilizing Effect of O-Glycosylation

May 11, 2017 - Department of Chemistry and Biochemistry and BioFrontiers Institute, University of Colorado, Boulder, Colorado 80303, United States. â€...
0 downloads 4 Views 1MB Size
Subscriber access provided by CORNELL UNIVERSITY LIBRARY

Article

Structural Insight into the Stabilizing Effect of O-Glycosylation Patrick K Chaffey, Xiaoyang Guan, Chao Chen, Yuan Ruan, Xingfeng Wang, Amy H Tran, Theo N Koelsch, Qiu Cui, Yingang Feng, and Zhongping Tan Biochemistry, Just Accepted Manuscript • Publication Date (Web): 11 May 2017 Downloaded from http://pubs.acs.org on May 12, 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.

Biochemistry 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 11

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

Biochemistry

Structural Insight into the Stabilizing Effect of O-Glycosylation Patrick K. Chaffey,1,‡ Xiaoyang Guan,1,‡ Chao Chen,2 Yuan Ruan,1 Xinfeng Wang,1 Amy H. Tran,1 Theo N. Koelsch,1 Qiu Cui,2 Yingang Feng,2,* and Zhongping Tan,1,* 1

Department of Chemistry and Biochemistry and BioFrontiers Institute, University of Colorado, Boulder, CO 80303, United States 2 Shandong Provincial Key Laboratory of Energy Genetics and CAS Key Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong Province 266101, China ABSTRACT: Protein glycosylation has been shown to have a variety of site-specific and glycan-specific effects, but so far the molecular logic that leads to such observations has been elusive. Understanding the structural changes that occur and being able to correlate those with the physical properties of the glycopeptide is a valuable step towards being able to predict how specific glycosylation patterns will affect the stability of glycoproteins. By systematically comparing the structural features of the O-glycosylated carbohydrate-binding module of a Trichoderma reesei-derived Family 7 cellobiohydrolase, we were able to develop a better understanding of the influence of O-glycan structure on the molecule’s physical stability. Our results indicate that the previously observed stabilizing effects of O-glycans come from the introduction of new bonding interactions to the structure and increased rigidity, while the decreased stability seemed to result from the impaired interactions and increased conformational flexibility. This type of knowledge provides a powerful and potentially general mechanism for improving the stability of proteins through glycoengineering.

INTRODUCTION Protein glycosylation is acknowledged as one of the most important post-translational modifications.1, 2 It involves the attachment of carbohydrates to amino acid side chains of proteins and is divided into categories based on the atom linking the carbohydrates to the protein. Of the many different types of protein glycosylation, N- and O-type are the most common and well-studied.3 In many cases, carbohydrate moieties have been shown to significantly alter the physical properties of proteins by stabilizing specific conformations, reducing selfassociation and biasing folding pathways.4, 5 From an engineering perspective, such physical effects could be exploited to improve industrial enzymes and therapeutic proteins.6, 7 Currently, designing any such improvements rationally is not feasible because the numerous structural factors that lead to the observable consequences of protein glycosylation are not well understood.8, 9 As a result, practical applications of protein glycosylation have been heavily restricted. A protein’s properties are largely determined by its threedimensional structure. To better understand the mechanisms by which carbohydrates can affect the properties of glycoproteins, it is important to gain detailed insights into their influence on the structures of glycoproteins. N-glycosylation is the most well studied form of glycosylation in this respect.10 Many studies on N-linked glycoproteins have established that the carbohydrates bias the conformation of the protein backbone towards more compact, more stable structures.11 This is particularly apparent in regions that are unstructured.8 Most of this effect seems to originate in the carbohydrate residues closest to the backbone, the so-called glycan core. A combination of hydrophobic and hydrophilic interactions between such core glycans and amino acids in the vicinity, which are indi-

vidually weak, can sum together to lead to the strong conformational shifts sometimes seen upon glycosylation.12

Figure 1. The amino acid sequence of the CBM with Oglycosylation sites R1-R3 indicated. The table displays both systematic and abbreviated names for each of the glycoforms studied. Proteolytic stability (half-life under thermolysin degradation) and thermal stability (melting temperatures measured by variable temperature CD) of each CBM variant are also included and come from our previous work with these molecules.13, 14 Below are the two O-glycan structures, 8 and 9, used in this work.

Studies of the effects of O-glycosylation on the structures of glycoproteins, on the other hand, are relatively rare; and most of the work in the area focuses on the ubiquitous, mostly unstructured mucin proteins.15 Mucins are a class of proteins that form a significant amount of the glycocalyx that surrounds and protects most cells. They are composed of many repeating,

ACS Paragon Plus Environment

Biochemistry

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

densely O-glycosylated domains, which together form a stable, extended structure in solution.16 While not as rigid as more typical secondary structural elements like α-helices or βsheets, this extended structure is considerably more organized than random coils or intrinsically disordered proteins.17 The glycans are critical to the structure of mucins, and removal of the O-glycans makes the proteins much more vulnerable to proteolytic degradation and much less structured.18 Although these studies provided some useful information, they only answer questions on the structural and functional features of O-linked glycans in one type of protein domain.19 In order to develop a better understanding of O-glycosylation, it is necessary to also study O-glycosylation of proteins with defined secondary and tertiary structures. In our ongoing efforts to study protein O-glycosylation, we have been systematically investigating the Family 1 carbohydrate-binding module (CBM) of the glycoside hydrolase Family 7 cellobiohydrolase from the cellulolytic fungus, Trichoderma reesei (TrCel7A), a key enzyme in the cellulosic biofuels industry.13, 14, 20 The CBM is a small domain responsible for the initially binding cellulose and bringing the catalytic domain of the cellulase enzyme into contact with the substrate. It has a well-defined structure and is small enough to conveniently determine structure-function relationships. Naturally, the CBM is glycosylated at Thr1, Ser3, and putatively at Ser14 (Fig. 1).13 All of these factors make the CBM an ideal model molecule to study the details of O-glycosylation. O-mannosylation of the CBM, which is the most common naturally occurring type of glycosylation in fungi, was found to make the small domain more resistant to both proteolytic attack and thermal denaturation.13 Most interestingly, we have found that even small changes in the glycosylation sites, gly-

Page 2 of 11

can structures and/or near-by amino acid side chains vary these effects.14 In particular, a Tyr residue near the glycosylation site Ser3 was identified as absolutely critical to the stabilizing influence of glycosylation. In addition, we verified that a nearby Gln was likewise important. These observations from our earlier work with the CBM prompted us to examine the structural differences among the representative glyco-variants (Fig. 1).13, 14 A better understanding of the domain’s structural features may help us to elucidate the molecular basis for the beneficial effects we have observed upon O-glycosylation of the CBM. Further down the road, this information may also provide useful guidance for rational glycoengineering of other proteins. RESULTS Design, preparation, and NMR study of CBM glycoforms. Previous work has shown that the core structures of glycans, both N- and O-types, have the largest impact on the physical properties of glycoproteins.14, 21 Since we were interested in studying the underlying structural changes that may lead to such changes in physical properties, we focused our study on only monosaccharide glycans (Fig. 1). The CBM glycoforms containing these glycans have relatively simple structures, which allowed for very reliable structural determination. With a focus on small glycans chosen, we designed the analogs for study to cover many of the important questions. Previous work with glycosylated CBM constructs has shown that, of the three possible natural glycosylation sites, Ser3 leads to the most robust increase in thermodynamic stability and protease resistance upon glycosylation (Fig. 1).13 CBMT1Manα 2,

Figure 2. Backbone overlay of NMR structures for each of the CBM variants. N indicates the N-terminus and C is the C-terminus. The glycan structures are shown in stick representation while backbones are ribbons. (A) CBM 1 (red) is compared with the previously determined unglycosylated structure (green) (PDB code: 2CBH). (B)-(E) compare unglycosylated CBM 1 (red) to the glycosylated CBM analogs 2-5. CBMS3Manα 3 is also shown rotated 90o to better show the relative positioning of the glycan moiety. (F)-(G) compare glycosylated CBMS3Manα 3 (blue) with the amino acid mutants CBMQ2A+S3Manα 6 and CBMQ2A+S3Manα 7.

ACS Paragon Plus Environment

Page 3 of 11

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

Biochemistry

CBMS3Manα 3, and CBMS14Manα 4 were therefore chosen to provide an explanation of how glycosylation at each of the three O-glycosylation sites can lead to drastically different physical properties, even when the glycan is of an identical size and composition, from the structural point of view. Furthermore, previously obtained results show that the attachment of a β-linked O-glucose (Glc) at the Ser3 had almost no effect on the stability of the CBM, unlike most of the other monosaccharides tested at the site and in stark contrast to α-linked O-mannose.14 We wished to better understand how the glycan structure and anomeric configuration of the linkage between glycan and peptide can lead to these observed physical changes. For this purpose, we chose to analyze and compare the structure of CBMS3Glcβ 5 with that of CBMS3Manα 3. Finally, mutation-based investigations into the amino acids surrounding Ser3 have revealed that both Gln2 and Tyr5 are very important to the stabilizing influence of glycosylation at this particular site.14 In order to gain a more fundamental insight into the role of these two amino acids, we chose to also gather structural data on the two glycosylated mutants: CBMQ2A+S3Manα 6 and CBMY5A+S3Manα 7, both of which are based on CBMS3Manα 3. Although enzymatic or chemo-enzymatic processes have been developed for hundreds of N-linked glycan structures22, 23 and even a few N-linked glycoproteins,24, 25 many of these are not applicable to O-linked glycopeptides and glycoproteins.26 Specific O-glycosylation patterns are not easily prepared using enzymatic approaches because of the lack of a well-defined consensus sequence for most O-glycosyltransferases. This complication makes it very difficult to ensure the selective preparation of particular glycoforms of a given Oglycopeptide.27 Chemical synthesis of glycoproteins, especially those containing only small O-glycans as used in this study,28-31 has been highly successful, and was therefore used to prepare the designed CBM glycoforms. Because glycosylation tends to significantly increase solubility, the generation of crystals from glycosylated proteins is often difficult.32, 33 We thus chose to use NMR to determine the structures of the designed CBM glycoforms. Previous work has established that the CBM does not form oligomers, which should ease the interpretation of NMR spectra. Additionally, NMR studies require far less optimization than crystallization for each new glycoform investigated.34 The NMR structure of each CBM variant was determined as described in the Methods section. A family of 20 accepted structures was derived for each variant (Fig. 2). Atomic coordinates of the structures and the chemical shift assignments have been deposited in the PDB and the BioMagResBank with accession codes 5X34 and 36050 (CBM 1), 5X35 and 36051 (CBMT1Manα 2), 5X36 and 36052 (CBMS3Manα 3), 5X37 and 36053 (CBMS14Manα 4), 5X38 and 36054 (CBMS3Glcβ 5), 5X39 and 36055 (CBMQ2A+S3Manα 6), and 5X3C and 36056 (CBMY5A+S3Manα 7). All structures were determined with high quality, and the statistics of restraints and structures are shown in the Supporting Information. Structural consequences of glycosylation at different sites. As expected, our NMR structure of the CBM 1 is very similar to the previously determined structure of the unglycosylated Cel7A CBM (PDB: 2CBH) (Fig. 2A).35 The minor differences between the two sets of structures are almost cer-

tainly attributable to changes in the exact approach and conditions used. With the obtained NMR structures, we first investigated the site-specific effects of O-mannosylation on the structural features of the CBM. Comparing the structures of CBMT1Manα 2, CBMS3Manα 3, and CBMS14Manα 4, which each contain a mono-mannose at a different site, there is a striking difference in the conformational variability of the linked glycan. Neither CBMT1Manα 2 nor CBMS14Manα 4 show their glycans converging into a single conformation (Fig. 2B and 2D). This means that the mannose residues in both glycoforms sample a wide range of conformational space and spend very little time in any particular orientation. In contrast, the mannose residue in CBMS3Manα 3 is relatively rigid and maintains a similar conformation for each of the 20 structures (Fig. 2C).

Figure 3. Amide NH bond chemical shift differences. Bars indicate the relative change for each of the 36 amide bonds in the backbone on the left as well as side chain amide shifts on the right (Gln or Asn, blue). (A) CBMT1Manα 2 and CBM 1, (B) CBMS3Manα 3 and CBM 1, (C) CBMS14Manα 4 and CBM 1, (D) CBMS3Glcβ 5 and CBM 1, (E) CBMQ2A+S3Manα 6 and CBM S3Manα 3, (F) CBMY5A+S3Manα 7 and CBMS3Manα 3.

Comparing the chemical shifts in the 15N HSQC spectra for unglycosylated CBM 1 and glycosylated CBMs 2-4, we found most of the differences caused by glycosylation are in two regions: residues 2-8 and 13-21 (Fig. 3). These results are not unexpected as previous findings have shown that O-

ACS Paragon Plus Environment

Biochemistry

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

glycosylation can perturb chemical shifts of residues that lie close to the glycosylation sites.36 For example, CBMT1Manα 2 showed large shift differences compared to CBM 1 for Gln2, Ser3, His4, and Tyr5, all of which are close to the site of glycosylation at Thr1 (Fig. 3A). Glycosylation at Ser3 also results in differences of chemical shift near the glycosylation site, from Gln2 to Cys8 (Fig. 3B). CBMS14Manα 4, on the other hand, displayed only slight changes in the chemical shifts of Tyr13, Ser14 and Gly15, which are all immediately adjacent to the glycosylation site at Ser14 (Fig. 3C). In addition, glycosylation at either Thr1 or Ser3 significantly affected chemical shifts of residues lying far away from the glycosylation sites in the primary sequence (Fig. 3). For example, CBMT1Manα 2 displays large changes in chemical shifts in the Thr17-Ser21 region of the sequence. CBMS3Manα 3 also has changes in this region, as seen in the chemical shifts of residues Gly15 through Ala20. The fact that these shifts were seen to change in CBM glyco-variants CBMT1Manα 2 and CBMS3Manα 3 relative to CBM 1, reflects the fact that this region is physically near the mannose glycans attached to Thr1 and Ser3 in the three-dimension structure of the peptide (Supporting Information). Overall, these data point to glycosylation in the Nterminal region (as found in glycoforms CBMT1Manα 2 and CBMS3Manα 3) having a more far-reaching effect on the peptide domain than glycosylation at other sites (as found in glycoform CBMS14Manα 4).

Page 4 of 11

CBMS3Manα 3 displays a large change in the chemical shift of a different residue, Gln2, as compared to the unglycosylated peptide (Fig. 3B). Such a large change is likely caused by the hydrogen bonds that are evident between mannose and Gln2 in the structure (Fig. 4A). These interactions were observed in all 20 of the lowest energy structures determined for each glycoform. Looking at the details of the interactions, we can clearly see the hydrogen bonds linking the glycan and peptide backbone. The side chain of Gln2 is seen to make hydrogen bonds with both the C4-O of the mannose ring and the side-chain oxygen of Ser3. In addition, the terminal CO of Gln2 is shown to be participating in a hydrogen bond with the OH of Tyr13’s side chain. This hydrogen bonding network may also contribute to the fairly rigid conformation of the mannose in this glycoform because this monosaccharide does not make significant contacts with any of the other nearby backbone groups. The signal for Gln34 was also observed to move significantly. Examining the three-dimensional structure reveals that Gln34 is located physically near Tyr5 and there is a hydrogen bond between the side-chain NH2 of Gln34 and CO of Tyr5. This raises the possibility that changes to Tyr5, which are caused by glycosylation at Ser3, are transferred to some extent to Gln34 resulting in the observed changes. Using the obtained NMR structures, we also investigated the possible changes that may be contributing to increased stability of certain CBM glyco-variants. Previous work by our group to characterize the CBM constructs CBMT1Manα 2, CBMS3Manα 3 and CBMS14Manα 4 showed that different glycosylation sites have different abilities to affect the proteolytic and thermal stability of the peptide (Fig. 1).13 Along with studies by other groups,37-39 our investigation suggested that the differences in the stability of the CBM variants are likely attributable to the site specific impact of glycans on conformational rigidity.14 Thus more stable variants tend to be more rigid. With the newly collected NMR structures revealed here, we could now test the plausibility of this explanation by comparing the root-mean-squared deviation (rmsd) values between the 20 structures of CBMs 1-4 and their average structures. Since all the structures were obtained under identical conditions and all calculations were done with identical protocols, the differences in rmsd values will reflect the differences in protein dynamics to some extent.40

Figure 4. Contacts made between the carbohydrate residue and peptide in (A) CBMS3Manα 3 and (B) CBMS3Glcβ 5. Right image is rotated 90o with respect to the top image.

When comparing the unglycosylated CBM 1 and glycosylated CBMT1Manα 2, the most notable single chemical shift change is the Ala20 NH signal. This could be due to the reduced interaction between Ala20 and residues at the Nterminus of the CBM. When Thr1 is not glycosylated, as is the case in CBM 1, the NH of Ala20 and the CO of Gln2 can be seen to form a hydrogen bond in the structure (Supporting Information).35 Although the same interaction appears present in many of the structures for CBMT1Manα 2, it seems possible that mannosylation of the near-by Thr1 residue causes a subtle shift in the conformation of Gln2, weakening this hydrogen bond and causing a large difference in chemical shift.

Figure 5. The root-mean squared deviation (rmsd) and transverse relaxation (T2) values of CBM variants. (A) Rmsd values (in angstroms Å) calculated for each CBM variant. Backbone rmsd is in white, heavy atom rmsd in grey. (B) Transverse relaxation (T2) times (in milliseconds ms) calculated for three regions of the NMR spectrum. Region 1 contains all the backbone amide NH bonds in each CBM variant (white). Region 2 contains methylene and methyl from most of the side chain groups (grey). Region 3 contains methyl groups from Ile 11 and Thr 1, 17, 23, and 24 (dark grey).

ACS Paragon Plus Environment

Page 5 of 11

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

Biochemistry

Global rmsd values were calculated using the molecular modeling software VMD41 through the RMSD Trajectory tool and the multiple sequence alignment and analysis tool MultiSeq.42 Because the glycan moiety is obviously more flexible in certain structures, the rmsd calculations for heavy atoms do not take into the account the differences in glycan position and only consider the side chain atoms of the CBM peptide. As shown Figure 5A, the values were 0.24 ± 0.04 Å for the backbone atoms of the unglycosylated CBM 1 and 0.42 ± 0.04 Å for all its heavy atoms (Supporting Information Table S1), the difference reflecting the greater flexibility of amino acid side chains compared to the protein backbone. CBMS3Manα 3 shows backbone and heavy atom rmsd values that are very similar to those of the unglycosylated CBM 1 (0.25 ± 0.04 Å for backbone and 0.41 ± 0.03 Å for heavy atoms). In contrast, the mannosylated variants CBMT1Manα 2 and CBMS14Manα 4 showed higher rmsd values: CBMT1Manα 2 had a backbone rmsd value of 0.32 ± 0.07 Å and a rmsd for all heavy atoms of 0.50 ± 0.06 Å; while CBMS14Manα 4 had values of 0.37 ± 0.05 Å and 0.57 ± 0.06 Å for backbone and heavy atoms respectively. For all four of these CBM glycoforms, the difference between backbone and heavy atom rmsd values were almost identical. This suggests that any flexibility introduced as a result of glycosylation equally impacts the backbone and side chains of the CBM. In order to get a more empirical look at the protein dynamics of the CBM glycopeptides, we were also able to measure the T2 for each of the structures.43 Without 15N labeling, the measurement of T2 for each individual amino acid residue is not feasible for sensitivity reasons.44 Instead, we utilized ordinary 1H relaxation experiments to measure T2 values for the entire glycopeptide structure. Three regions of the NMR spectrum were used for this purpose. As described in Figure 5, Region 1 covers the backbone amide NH bonds of the molecules. Region 2 covers the methylene and methyl groups of most of the side chain groups for the peptide. Finally, region 3 includes the methyl groups of Ile and Thr residues. Since they represent signals from across the entire sequence, these T2 values are an experimental measure of the overall backbone and side chain dynamics.45 This T2 data largely follows the same trend as that of the rmsd calculations. Both CBMT1Manα 2 and CBMS14Manα 4 show significant increases in the relaxation times, which translates to a faster rotational motion and conformational exchange of the backbone in these glycoforms.43 Compared to CBM 1, CBMS3Manα 3 actually had a decreased amide NH T2 value. This reflects a more rigid backbone structure as a result of glycosylation at Ser3, a property that was not seen to the same extent after glycosylation at either Thr1 or Ser14 as discussed above. The influence of glycan structures on the structures of the CBM variants. Changing the glycan from α-linked mannose to β-linked glucose significantly diminishes the effects of glycosylation on the physical properties of the CBM.14 This motivated us to test changes to the especially stable structure of CBMS3Manα 3. Interestingly, like the α-Man of CBMS3Manα 3, the β-Glc moiety also occupies a single conformation, albeit with slightly greater flexibility (Fig. 2E). Again, hydrogen bonds probably contribute to the relatively small conformational space

sampled by the β-Glc: they seem to link the C2-OH of the βGlc and the backbone CO of His4 as well as the C6-OH of βGlc and the backbone CO of Thr1 (Fig. 4B). Compared to the mannose of CBMS3Manα 3, the glucose of CBMS3Glcβ 5 takes on a much more angled position with respect to the Gln2 sidechain, which has two effects. First, the contacts seen between mannose and Gln2’s side chain (Fig. 4A) are absent in CBMS3Glcβ 5 (Fig. 4B), probably due to the greater distance between the hydroxyl groups of glucose and the terminal amide of Gln2. The Gln2 side chain is rotated compared to that of CBMS3Manα 3, such that the terminal NH forms a hydrogen bond with the side chain of Tyr13. This conformation also allows the CO of Gln2’s side chain to form a bond with the backbone NH of Ser3, another difference between CBMS3Manα 3 and CBMS3Glcβ 5 (Fig. 4). In addition, because of the angle of the glucose ring, both the C2-OH and C6-OH groups are brought into closer proximity to the backbone and can now form hydrogen bonds with the backbone CO groups of His4 and Thr1, respectively (Fig. 4B). When glycosylation at Ser3 is β-Glc, instead of α-Man, there are very similar changes in the chemical shifts near the glycosylation site and throughout the glycopeptide (Fig. 3D). The magnitude of most of these shift changes is significantly lower, however. Specifically, the signals of Thr1 and His4, which are shown to be involved in hydrogen bonds with the glucose residue, are changed very little from those of the unglycosylated CBM. The largest shifts compared to those of CBM 1 are in the signals of Gln2. Looking at the structure, the side chain of the terminal amide of Gln2 appears to be participating in hydrogen bonds with both the OH of nearby-by Tyr13 and the CO of Ser3 (Fig. 4B and Supporting Information). These interactions are not present in the unglycosylated CBM 1 and could explain the chemical shift differences observed here. Looking at the rmsd values for CBMS3Glcβ 5, we have 0.32 ± 0.09 Å for backbone and 0.48 ± 0.08 Å for heavy atoms (Fig. 5A). The slight increase in backbone flexibility is of a similar magnitude as that seen for CBMT1Manα 2. This shows that the anomeric and stereochemical layout of the carbohydrate also affects the physical nature and conformation of the peptide in addition to the site of glycosylation. The increase in heavy atom rmsd of β-Glc at Ser3 as opposed to αMan at Ser3 is significant, but not as large as the similar increase when moving the α-Man from Ser3 to Ser14. Transverse relaxation times of this glycoform tell a similar story. The T2 value for CBMS3Glcβ 5 is higher than that of CBMS3Manα 3. Thus, unlike α-Man at this position, the βGlc monomer does less to change the conformational exchange rate of the backbone amide bonds and leads to a similar amount of flexibility as the unglycosylated peptide. The influence of near-by amino acid side chains on the structures of the CBM variants. Given the importance of the local amino acids to the stability of CBMS3Manα 3, we next characterized how the mutations affect its structure. As shown in Figure 2F, without the Gln2 side chain to anchor the α-Man to the peptide, the glycan is significantly more flexible. The chemical shift differences are significant between the Q2A mutant CBMQ2A+S3Manα 6 and CBMS3Manα 3. As expected, the most significant differences are around the Q2A mutation, suggesting that the removal of the side chain of Gln2 has a strong effect on the neighboring amide bonds. We

ACS Paragon Plus Environment

Biochemistry

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

also observed changes in the Thr17-Cys25 and Ser33-Leu36 regions, which are either physically near the N-terminal region of the sequence in the three-dimensional structure of the domain or interact with the N-terminus through hydrogen bond network (Supporting Information). The chemical shift change of Ala20 is especially large. This could be due to the reduced direct interaction between the backbone of Ala20 and that of Gln2. The most significant structural change is that the methyl group of Ala2 is more buried than Gln2 in other CBMs. This rotates the side chain of Cys35 such that the χ1 torsion angle is in the trans conformation instead of the gauche(-) conformation seen in other structures. Additionally, the disulfide bond between Cys19 and Cys35 adopts a trans conformation unlike other CBMs where a cis conformation is seen (Supporting Information). The reduced intramolecular interactions are also reflected in the calculated rmsd values, which are 0.31 ± 0.06 Å for the backbone and 0.49 ± 0.06 Å for the heavy atoms. These values are very similar to those of CBMT1Manα 2 and CBMS3Glcβ 5, and like both CBMT1Manα 2 and CBMS3Glcβ 5, the proteolytic and thermal stabilities of CBMQ2A+S3Manα 6 are low. Interestingly, the T2 values for CBMQ2A+S3Manα 6 do not reflect this conformational flexibility in the glycan, and are quite similar to those of the much more stable CBMS3Manα 3. These observations do not necessarily contradict one another as previous work has linked T2 values to not only conformational rotation at the microsecond to millisecond time scale but also the rate of rotational diffusion for the molecule as a whole and low-affinity self-association.46, 47, 48 These properties would be expected to be affected by the Gln-to-Ala mutation and affect one another, as molecular rotation is known to depend on size. Tyrosine 5 is another N-terminal residue that is critical to the stabilizing effect of the Ser3 Man. Its mutation had been previously shown to totally ameliorate the stabilizing influence of the glycan on the peptide structure. Therefore, we decided to also obtain structural characterization for the Y5A mutant carrying a single mannose at Ser3. In the NMR structure obtained here (Fig. 2G), we can clearly see that, without the side chain of Tyr5, the glycan does not converge into a single conformation. These changes may result from the removal of a His4-Tyr5 aromatic stacking interaction, which can be seen in the other structures reported here (Supporting Information) and has been observed by others.49 Such a pi-pi stacking interaction could act to stabilize the domain’s conformation. However, in mutant CBMY5A+S3Manα 7, with such an interaction absent, His4 shifts into a significantly different conformation and subsequently, leads to changes further down the peptide backbone (Supporting Information). The chemical shift differences between the CBMY5A+S3Manα 7 analog and CBMS3Manα 3 are also telling (Fig. 3F). The largest difference between the two occurs in the N-terminal region, near the mutation. The differences are much greater than the analogous changes that occur upon mutation of Gln2. This seems to reflect the greater differences that the Y5A mutation causes to the N-terminal region of the structure. Particularly interesting is the large difference in HE22 shift of Gln2 comparing CBMY5A+S3Manα 7 and CBMS3Manα 3. This is the hydrogen involved in a hydrogen bond between the Ser3 α-Man glycan and the amide side chain of Gln2. It is also worth noting that the aromatic ring of Tyr5 is almost certainly responsible for local shielding

Page 6 of 11

and deshielding of adjacent protons. Removing this aromatic ring would be expected to alter the observed chemical shifts for such protons, independent of any other structural changes that may occur in the vicinity. These observations which presumably reflect weakened intramolecular interactions are also echoed in the rmsd values calculated for the structure, 0.37 ± 0.07 Å for backbone atoms and 0.55 ± 0.06 Å for heavy atoms as well as in the T2 relaxation value. As shown in Figure 5, this glycoform had the highest transverse relaxation time of any of the seven glycoforms characterized here. From these measurements, it is apparent that the loss of the pi-pi interaction between His4 and Tyr5 leads to significantly more mobility in the backbone of the CBM. DISCUSSION Glycosylation is the most common posttranslational modification of proteins that are secreted from cells.1, 2 Both O- and N-glycosylation can enhance protein physical stability through carbohydrate-protein interactions.11 If general, such effects hold much promise for developing industrial enzymes and protein therapeutics with improved properties.32 However, because the molecular basis for these effects is incompletely understood, rational manipulation of the properties of proteins through glycoengineering has not be achieved. Prior to this work, examining the structural details of several closelyrelated N-glycosylated proteins had been demonstrated to be a uniquely powerful means of investigating the molecular details that underpin the effects of glycosylation and might lead to clearer glycoengineering guidelines in the future.12, 38 Here, we applied a similar strategy to investigate the stabilizing influence of O-linked glycans. By using NMR, we were able to carry out an in-depth study of 6 different CBM glyco-variants. Furthermore, our previous work with these glycoforms put us in a unique position to correlate the transformed structural features to physical stability.13, 14 Our structures show that, depending on the attachment site, an O-linked glycan will interact to different degrees with the local amino acids. Depending on the nature of these contacts, the glycan moiety can adopt either a single rigid conformation or is free to adopt many conformations over the time frame of the NMR experiments. In particular, only when the glycan was attached to Ser3, as in CBMS3Manα 3 and CBMS3Glcβ 5, were the glycans seen to mostly occupy a single conformation. Presumably, it is the hydrogen bonds identified between the mannose of CBMS3Manα 3 or the glucose of CBMS3Glcβ 5 and near-by amino acid side chains can help these two sugars to occupy a single conformation on the NMR time-scale. These same bonding interactions would be absent at the other two sites, which could help to explain how glycosylation at those failed to give a convergent glycan conformation. Although the local interactions between the glycan and peptide may be necessary, they are not sufficient for increasing the stability of the glycosylated molecule. Comparing CBMS3Manα 3 and CBMS3Glcβ 5, we were able to see that while glycans in both molecules seemed fairly rigid, the stability of CBMS3Manα 3 was far higher than CBMS3Glcβ 5. This could be the result of interactions formed between the αMan glycan and Gln2 of CBMS3Manα 3 being propagated throughout the rest of the backbone, strengthening the global network of intramolecular interactions and leading to a de-

ACS Paragon Plus Environment

Page 7 of 11

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

Biochemistry

crease in the CBM’s backbone dynamics. Lower backbone flexibility should allow the CBM to maintain its native structure under higher temperature, while simultaneously inhibiting the peptide from adopting optimal conformations for protease cleavage, thus increasing both its thermal and proteolytic stability. In the case of CBMS3Glcβ 5, although the sugar moiety forms two additional hydrogen bonds with the peptide backbone, these interactions may only affect CBM backbone dynamics locally, in the N-terminus, and thus the glucosylation of Ser3 cannot significantly improve the stability of the entire CBM. This suggests that, although glycans may form many kinds of interactions with proteins, there are specific, key intramolecular contacts that must be present to significantly change the stability of a protein.

Figure 6. Correlation of the change in melting temperature (∆Tm) and change in half-life during thermolysin degradation (∆t1/2), and (A) the change in rmsd and (B) in T2 (Region 1) upon glycosylation. Data points represent differences between the glyco-variants and the unglycosylated CBM 1. The Pearson’s correlation coeffi-

cient values (r) are inscribed on each planar projection to describe the strength of each correlation. A positive value means that the two variables change in the same direction and a negative value indicates changes in opposite directions. The larger the magnitude of the value, the stronger the correlation between the two variables.

Our results also suggest that certain key molecular interactions must be persevered in order to make a molecule more stable. This finding was illustrated by variants CBMQ2A+S3Manα 6, which has a Gln2 to Ala (Q2A) mutation, and CBMY5A+S3Manα 7, which has a Tyr5 to Ala (Y5A) mutation. Gln2 is a key residue in the intramolecular interaction network. Removing the side chain of Gln2 in CBMQ2A+S3Manα 6 would obviously lead to the loss of interactions participated in by Gln2’s side chain in CBMS3Manα 3 and we observed that this resulted in a much less stable glycosylated peptide. Tyr5 is known to be part of an important pi-pi interaction with the neighboring histidine residue. Mutation of Tyr5 to Ala disrupted this key interaction and we saw this lead to global backbone changes including abolishing interactions between the Man glycan and Gln2. These changes resulted in a severe decrease in stability. Overall, our study indicates that there is a rough link between the decreased dynamics and increased physical stability (Fig. 6). However, because stability of large molecules like proteins is a complicated phenomenon and is controlled by many factors, only one of which is conformational flexibility,50 it is expected that some exceptions to this simple correlation be found. For example, CBMS14Manα 4, which was shown to have a more flexible backbone, was actually more stable than CBMS3Glcβ 5. CONCLUSIONS By comparing the NMR structures of glycosylated CBMs with systematic variations in their glycans structures, amino acid sequences, and glycosylation sites, we were able to understand how protein O-glycosylation affects the conformational changes of glycoproteins in more detail. From the results, we can conclude that differently glycosylated CBM variants form unique intramolecular interactions, which could be a consequence of the changed points of contact between the various glycan moieties and the local amino acids. Changes to the backbone dynamics of each variant follow in turn from these subtle differences in intramolecular interactions. These new structural data, together with our previous results on the physical stability of these variants, suggest that Oglycosylation stabilizes the structures by, at least partially, increasing the molecules’ rigidity through the formation of interactions with key residues. Our study clarifies the molecular basis for the increased physical stability that can occur as a direct result of Oglycosylation. Although many knowledge gaps remain and further work is necessary to confirm the generality of our findings, this important first step is expected to greatly facilitate future studies of protein glycosylation and the rational deployment of glycosylation to engineer proteins with tailored properties for a variety of uses. METHODS

ACS Paragon Plus Environment

Biochemistry

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

General Procedure for the Preparation of CBM Glycoforms. The synthesis and characterization were carried out following the procedure previously developed in our laboratory.13, 14 Briefly, standard Fmoc-based solid-phase peptide synthesis (SPPS) was used to construct each CBM glycopeptide variant. Glycans were introduced via synthetically prepared glycoamino acid building blocks using coupling conditions identical to those of non-glycosylated building blocks except for a longer coupling time. During SPPS, all glycan hydroxyl groups were protected with acetyl groups. After SPPS was complete, the glycopeptide was deprotected and cleaved from the resin using a TFA/TIS/H2O cocktail. Acetyl groups protecting the glycans were removed with a short hydrazine treatment and subsequent folding under optimized pH- and redox-buffered conditions gave the correct disulfide pairing and 3D structure.13, 14 Purification by HPLC yielded the final samples in high purity. Determination of NMR Solution Structures of CBM Variants. Each sample of about 5 mg was divided into two parts: one part being dissolved in an acetate buffer, pH 5.0, in 90% H2O and 10% D2O, while the other part being dissolved the same buffer in 100% D2O. All NMR experiments were recorded on Bruker Avance III 600 MHz spectrometer equipped with a triple-resonance TCI cryoprobe and zgradient. Spectral processing was carried out using NMRPipe and was analyzed by NMRViewJ software.51 The chemical shifts were referenced to an internal standard: 0.01% (w/v) sodium 2,2-dimethylsilapentane-5-sulfonate (DSS). The 1H chemical shift assignments of each CBM variant were obtained from 2D 1H-1H DQF-COSY, 1H-1H TOCSY, 1 H-1H NOESY (with mixing time of 200 ms) experiments, referring the previously published assignments of the CBM. Most of the 13C and 15N chemical shifts were assigned from the 2D 1H-13C HSQC, 1H-13C HSQC-TOCSY, and 1H-15N HSQC spectra. The unambiguous assignments of the chemical shifts of the sugar ring carbon and hydrogen atoms were achieved through the combined use of the 2D 1H-13C HSQC, H2BC and 1H-1H COSY experiments.51 The NMR structure of each CBM variant was determined largely based on the distance restraints derived from the 2D 1 H-1H NOESY spectra of its two sample parts. The NOE peak assignments were obtained semi-automatically with the program SANE. The backbone torsion angles predicted from 1H, 13 C and 15N chemical shifts by TALOS-N analysis were also used as constraints for structure calculation. Partial stereo assignments of methylene hydrogens were obtained by the analysis of the intra-residue NOE patterns, which generated the side chain χ1 and χ2 torsion angle restraints for structure refinements. The topology and parameter files for the sugars were created by modifying the original files. 100 structures were initially calculated by the software CNS and 50 lowestenergy structures were selected and subjected to refinement in explicit water using RECOODScript.52 After the refinement, 20 lowest-energy structures were selected for violation and structure analysis. After multiple iterative calculations, the 20 lowest-energy structures without violations greater than 0.2 Å for distance restraints and 5 degree for torsion angle restraints were used as final structures for representation and analysis. The quality of these structures was analyzed using the programs PyMOL (Schrödinger, LCC), PROCHECK-NMR,53 and WHATCHECK.54 PyMOL was used for visualization of the structures.

Page 8 of 11

The chemical shift differences (CSD) were calculated using the following formula, CSD = (δ HN ) 2 + (

δN

) 2 where δHN 6 and δN are the changes of 1HN and 15N chemical shifts, respectively. The T2 measurements were performed using a Carr-PurcellMeiboom-Gill sequence with a pre-saturation water suppression. The data were processed and analyzed using the T1/T2 module of the program Topspin 3.2 (Bruker Biospin).

ASSOCIATED CONTENT Supporting Information. Experimental procedures, spectroscopic and analytical data. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * [email protected]; [email protected]

Notes The authors declare no competing financial interest.

Author Contributions All authors have given approval to the final version of the manuscript. ‡These authors contributed equally.

ACKNOWLEDGMENT We would like to thank the University of Colorado Boulder (Start-up fund), the NSF CAREER Award (Grant number: CHE1454925), the National Natural Science Foundation of China (Grant numbers: 31670735 and 31661143023) and Chinese Government Scholarship (Grant number: 201604910035) for their support during the course of this study.

ABBREVIATIONS CBM, carbohydrate-binding module; rmsd, root-mean squared deviation; T2, transverse relaxation.

REFERENCES (1) Walsh, C. T., Garneau-Tsodikova, S., and Gatto, G. J., Jr. (2005) Protein posttranslational modifications: the chemistry of proteome diversifications, Angew. Chem. Int. Ed. Engl. 44, 73427372. (2) Prabakaran, S., Lippens, G., Steen, H., and Gunawardena, J. (2012) Post-translational modification: nature's escape from genetic imprisonment and the basis for dynamic information encoding, Wiley Interdiscip Rev Syst Biol Med 4, 565-583. (3) Varki, A. (2009) Essentials of glycobiology, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (4) Varki, A. (1993) Biological roles of oligosaccharides: all of the theories are correct, Glycobiology 3, 97-130. (5) Varki, A. (2016) Biological Roles of Glycans, Glycobiology. (6) Sinclair, A. M., and Elliott, S. (2005) Glycoengineering: the effect of glycosylation on the properties of therapeutic proteins, J. Pharm. Sci. 94, 1626-1635. (7) Greene, E. R., Himmel, M. E., Beckham, G. T., and Tan, Z. (2015) Glycosylation of cellulases: Engineering better enzymes for biofuels, Adv. Carbohydr. Chem. Biochem. 72, 63-112. (8) Shental-Bechor, D., and Levy, Y. (2009) Folding of glycoproteins: toward understanding the biophysics of the glycosylation code, Curr. Opin. Struct. Biol. 19, 524-533.

ACS Paragon Plus Environment

Page 9 of 11

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

Biochemistry

(9) Hebert, D. N., Lamriben, L., Powers, E. T., and Kelly, J. W. (2014) The intrinsic and extrinsic effects of N-linked glycans on glycoproteostasis, Nat. Chem. Biol. 10, 902-910. (10) Nettleship, J. E. (2012) Structural Biology of Glycoproteins, INTECH Open Access Publisher. (11) Imperiali, B., and O'Connor, S. E. (1999) Effect of N-linked glycosylation on glycopeptide and glycoprotein structure, Curr. Opin. Chem. Biol. 3, 643-649. (12) Price, J. L., Culyba, E. K., Chen, W., Murray, A. N., Hanson, S. R., Wong, C. H., Powers, E. T., and Kelly, J. W. (2012) N-glycosylation of enhanced aromatic sequons to increase glycoprotein stability, Biopolymers 98, 195-211. (13) Chen, L., Drake, M. R., Resch, M. G., Greene, E. R., Himmel, M. E., Chaffey, P. K., Beckham, G. T., and Tan, Z. (2014) Specificity of O-glycosylation in enhancing the stability and cellulose binding affinity of Family 1 carbohydrate-binding modules, Proc. Natl. Acad. Sci. U S A 111, 7612-7617. (14) Guan, X., Chaffey, P. K., Zeng, C., Greene, E. R., Chen, L., Drake, M. R., Chen, C., Groobman, A., Resch, M. G., Himmel, M. E., Beckham, G. T., and Tan, Z. (2015) Molecular-scale features that govern the effects of O-glycosylation on a carbohydrate-binding module, Chem. Sci. 6, 7185-7189. (15) Barb, A. W., Borgert, A. J., Liu, M., Barany, G., and Live, D. (2010) Intramolecular glycan-protein interactions in glycoproteins, Methods Enzymol. 478, 365-388. (16) Hang, H. C., and Bertozzi, C. R. (2005) The chemistry and biology of mucin-type O-linked glycosylation, Bioorg. Med. Chem. 13, 5021-5034. (17) Barchi, J. J. (2013) Mucin-type glycopeptide structure in solution: Past, Present, and Future, Biopolymers 99, 713-723. (18) Bergstrom, K. S., and Xia, L. (2013) Mucin-type O-glycans and their roles in intestinal homeostasis, Glycobiology 23, 10261037. (19) Steen, P. V. d., Rudd, P. M., Dwek, R. A., and Opdenakker, G. (1998) Concepts and principles of O-linked glycosylation, Crit. Rev. Biochem. Mol. Biol. 33, 151-208. (20) Happs, R. M., Guan, X., Resch, M. G., Davis, M. F., Beckham, G. T., Tan, Z., and Crowley, M. F. (2015) Oglycosylation effects on family 1 carbohydrate-binding module solution structures, FEBS J. 282, 4341-4356. (21) Hanson, S. R., Culyba, E. K., Hsu, T. L., Wong, C. H., Kelly, J. W., and Powers, E. T. (2009) The core trisaccharide of an N-linked glycoprotein intrinsically accelerates folding and enhances stability, Proc. Natl. Acad. Sci. U S A 106, 3131-3136. (22) Wang, Z., Chinoy, Z. S., Ambre, S. G., Peng, W., McBride, R., de Vries, R. P., Glushka, J., Paulson, J. C., and Boons, G. J. (2013) A general strategy for the chemoenzymatic synthesis of asymmetrically branched N-glycans, Science 341, 379-383. (23) Li, L., Liu, Y., Ma, C., Qu, J., Calderon, A. D., Wu, B., Wei, N., Wang, X., Guo, Y., Xiao, Z., Song, J., Sugiarto, G., Li, Y., Yu, H., Chen, X., and Wang, P. G. (2015) Efficient chemoenzymatic synthesis of an N-glycan isomer library, Chem. Sci. 6, 5652-5661. (24) Wang, L. X., and Davis, B. G. (2013) Realizing the promise of chemical glycobiology, Chem. Sci. 4, 3381-3394. (25) Wildt, S., and Gerngross, T. U. (2005) The humanization of N-glycosylation pathways in yeast, Nat. Rev. Microbiol. 3, 119128. (26) McArthur, J. B., and Chen, X. (2016) Glycosyltransferase engineering for carbohydrate synthesis, Biochem. Soc. Trans. 44, 129-142. (27) Gamblin, D. P., Scanlan, E. M., and Davis, B. G. (2009) Glycoprotein synthesis: an update, Chem. Rev. 109, 131-163. (28) Bertozzi, C. R., and Kiessling, L. L. (2001) Chemical glycobiology, Science 291, 2357-2364.

(29) Boltje, T. J., Buskas, T., and Boons, G. J. (2009) Opportunities and challenges in synthetic oligosaccharide and glycoconjugate research, Nat. Chem. 1, 611-622. (30) Krasnova, L., and Wong, C. H. (2016) Understanding the chemistry and biology of glycosylation with glycan synthesis, Annu. Rev. Biochem. 85, 599-630. (31) Fernandez-Tejada, A., Brailsford, J., Zhang, Q., Shieh, J. H., Moore, M. A., and Danishefsky, S. J. (2015) Total synthesis of glycosylated proteins, Top. Curr. Chem. 362, 1-26. (32) Sola, R. J., and Griebenow, K. (2009) Effects of glycosylation on the stability of protein pharmaceuticals, J. Pharm. Sci. 98, 1223-1245. (33) Tams, J. W., Vind, J., and Welinder, K. G. (1999) Adapting protein solubility by glycosylation. N-glycosylation mutants of Coprinus cinereus peroxidase in salt and organic solutions, Biochim. Biophys. Acta 1432, 214-221. (34) Mer, G., Hietter, H., and Lefevre, J. F. (1996) Stabilization of proteins by glycosylation examined by NMR analysis of a fucosylated proteinase inhibitor, Nat. Struct. Biol. 3, 45-53. (35) Kraulis, J., Clore, G. M., Nilges, M., Jones, T. A., Pettersson, G., Knowles, J., and Gronenborn, A. M. (1989) Determination of the three-dimensional solution structure of the C-terminal domain of cellobiohydrolase I from Trichoderma reesei. A study using nuclear magnetic resonance and hybrid distance geometry-dynamical simulated annealing, Biochemistry 28, 7241-7257. (36) Grinstead, J. S., Koganty, R. R., Krantz, M. J., Longenecker, B. M., and Campbell, A. P. (2002) Effect of glycosylation on MUC1 humoral immune recognition: NMR studies of MUC1 glycopeptide-antibody interactions, Biochemistry 41, 9946-9961. (37) Rudd, P. M., Joao, H. C., Coghill, E., Fiten, P., Saunders, M. R., Opdenakker, G., and Dwek, R. A. (1994) Glycoforms modify the dynamic stability and functional activity of an enzyme, Biochemistry 33, 17-22. (38) O'Connor, S. E., Pohlmann, J., Imperiali, B., Saskiawan, I., and Yamamoto, K. (2001) Probing the effect of the outer saccharide residues of N-linked glycans on peptide conformation, J. Am. Chem. Soc. 123, 6187-6188. (39) Yu, H., and Huang, H. (2014) Engineering proteins for thermostability through rigidifying flexible sites, Biotechnol. Adv. 32, 308-315. (40) Yang, L. W., Eyal, E., Chennubhotla, C., Jee, J., Gronenborn, A. M., and Bahar, I. (2007) Insights into equilibrium dynamics of proteins from comparison of NMR and X-ray data with computational predictions, Structure 15, 741-749. (41) Humphrey, W., Dalke, A., and Schulten, K. (1996) VMD: visual molecular dynamics, J. Mol. Graph. 14, 33-38, 27-38. (42) Roberts, E., Eargle, J., Wright, D., and Luthey-Schulten, Z. (2006) MultiSeq: unifying sequence and structure data for evolutionary analysis, BMC Bioinformatics 7, 382. (43) Kleckner, I. R., and Foster, M. P. (2011) An introduction to NMR-based approaches for measuring protein dynamics, Biochim. Biophys. Acta 1814, 942-968. (44) Gardner, K. H., and Kay, L. E. (1998) The use of 2H, 13C, 15N multidimensional NMR to study the structure and dynamics of proteins, Annu. Rev. Biophys. Biomol. Struct. 27, 357-406. (45) Fischer, M. W. F., Majumdar, A., and Zuiderweg, E. R. P. (1998) Protein NMR relaxation: theory, applications and outlook, Prog. Nucl. Magn. Reson. Spectrosc. 33, 207-272. (46) Kroenke, C. D., Loria, J. P., Lee, L. K., Rance, M., and Palmer, A. G. (1998) Longitudinal and transverse 1H−15N dipolar/15N chemical shift anisotropy relaxation interference:  Unambiguous determination of rotational diffusion tensors and chemical exchange effects in biological macromolecules, J. Am. Chem. Soc. 120, 7905-7915.

ACS Paragon Plus Environment

Biochemistry

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

(47) Pfuhl, M., Chen, H. A., Kristensen, S. M., and Driscoll, P. C. (1999) NMR exchange broadening arising from specific low affinity protein self-association: analysis of nitrogen-15 nuclear relaxation for rat CD2 domain 1, J. Biomol. NMR 14, 307-320. (48) Ishima, R., and Torchia, D. A. (2000) Protein dynamics from NMR, Nat. Struct. Biol. 7, 740-743. (49) Mattinen, M. L., Kontteli, M., Kerovuo, J., Linder, M., Annila, A., Lindeberg, G., Reinikainen, T., and Drakenberg, T. (1997) Three-dimensional structures of three engineered cellulose-binding domains of cellobiohydrolase I from Trichoderma reesei, Protein Sci. 6, 294-303. (50) Karshikoff, A., Nilsson, L., and Ladenstein, R. (2015) Rigidity versus flexibility: the dilemma of understanding protein thermal stability, FEBS J. 282, 3899-3917. (51) Delaglio, F., Grzesiek, S., Vuister, G. W., Zhu, G., Pfeifer, J., and Bax, A. (1995) NMRPipe: a multidimensional spectral processing system based on UNIX pipes, J. Biomol. NMR 6, 277293.

Page 10 of 11

(52) Nederveen, A. J., Doreleijers, J. F., Vranken, W., Miller, Z., Spronk, C. A., Nabuurs, S. B., Guntert, P., Livny, M., Markley, J. L., Nilges, M., Ulrich, E. L., Kaptein, R., and Bonvin, A. M. (2005) RECOORD: a recalculated coordinate database of 500+ proteins from the PDB using restraints from the BioMagResBank, Proteins 59, 662-672. (53) Laskowski, R. A., Rullmannn, J. A., MacArthur, M. W., Kaptein, R., and Thornton, J. M. (1996) AQUA and PROCHECK-NMR: programs for checking the quality of protein structures solved by NMR, J. Biomol. NMR 8, 477-486. (54) Hooft, R. W., Vriend, G., Sander, C., and Abola, E. E. (1996) Errors in protein structures, Nature 381, 272.

ACS Paragon Plus Environment

Page 11 of 11

Biochemistry

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 ACS Paragon Plus Environment

11