Surface Binding Site

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• Authors: ^^^Birte Svensson^^^ and ^^^Darrell Cockburn^^^
• Responsible Curators: ^^^Birte Svensson^^^ and ^^^Spencer Williams^^^

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Surface Binding Sites

Figure 1. The barley α-amylase 1 in complex with maltoheptaose PDB ID 1rp8 [1]. Several of the key SBS residues are shown highlighted in yellow, while the maltoheptaose molecules are shown in orange. Note the relatively large distance from the active site, which is a common aspect of these sites.

A surface (or secondary) binding site (SBS) is a ligand binding site observed on the catalytic module of an enzyme, but outside of the active site itself (see Figure 1). For recent reviews on this topic, please see [2, 3, 4].

Detection and Occurrence

SBSs have been observed in the crystal structures of approximately 50 carbohydrate active enzymes, with about half of these enzymes belonging to the GH13 family (Table 1). Typically the enzymes found to possess one or more SBSs are active on polysaccharides, suggesting that SBSs are adaptations for dealing with longer substrates. X-ray crystallography has been the main method of detecting SBSs; however, NMR [5] and chemical labeling [6] have also been used in the detection of these features. Examination of the SBS containing enzymes show that they frequently co-occur with carbohydrate-binding modules (CBMs), suggesting that these two methods of binding to a substrate are largely complementary rather than redundant [2]. In one example in particular, SusG from Bacteroides thetaiotaomicron, both a CBM and an SBS were found to contribute to binding to starch granules [7].

Roles of SBSs in Enzyme Function

Detailed analyses of SBSs have only been carried out in a few cases, however, in each of these cases they have been found to be important for the function of the enzyme. These and other hypothesized roles have been recently reviewed [2, 3, 4]. In general the proposed roles of SBSs can be summarized as: i) serving as an extension of the active site, guiding a substrate strand to the active site or maintaining a hold on the strand to allow processivity, ii) acting as an allosteric regulator, with binding at the SBS affecting the properties of the active site, iii) serving as a pseudo-CBM, by targeting the enzyme to the substrate, anchoring the enzyme to the cell wall or disrupting the substrate (see the carbohydrate-binding modules page for more details on their functional roles). As an illustrative example, the two SBSs of the barley α-amylase 1(named SBS1 and SBS2) [1] seem to fall into categories i) and iii). SBS1 is particularly important for the binding of the enzyme to starch granules [8], while SBS2 is more important for the enzyme’s activity on amylopectin, lowering the apparent KM for this substrate [9]. A good example of ii) is seen in the amylomaltase from Thermus aquaticus, where binding to the SBS changes the active site, thereby altering the substrate profile of the enzyme [10].

Studying SBSs

The study of SBSs is often complicated by the presence of multiple binding sites in a given enzyme due to the frequent occurrence of multiple SBSs in a given enzyme, binding in the active site or the presence of a CBM. Various techniques have been used to isolate SBSs for individual study such as the use of mutations and substrates that do not penetrate the active site [8] or the use of covalent inhibitors to block the active site [5, 11]. A variety of techniques have proven useful for studying SBSs, including surface plasmon resonance, isothermal titration calorimetry, affinity electrophoresis and adsorption assays (the use of these techniques and others is summarized in [2]).

Table 1: Glycoside hydrolase enzyme families for which an enzyme with an SBS has been identified.
Family	# of Enzymes as of 2015-02-17	Example Structure	Reference(s)
GH1	2	1uyq	Unpublished
GH5	1	2pc8	[12]
GH8	1	2b4f	[13]
GH10	2	1goq	[14, 15]
GH11	3	2qz3	[5, 16]
GH13	23	1rp8	[1, 2, 3]
GH14	1	1b9z	[17]
GH15	1	2f6d	[18]
GH16	1	1urx	[19]
GH19	1	3cql	[20]
GH27	1	3hg2	[21]
GH31	1	3nqq	Unpublished
GH34	1	1mwe	[22]
GH57	1	3n98	[23]
GH63	1	3c67	[24]
GH77	1	1esw	[25]

References

1. Robert X, Haser R, Mori H, Svensson B, and Aghajari N. (2005). Oligosaccharide binding to barley alpha-amylase 1. J Biol Chem. 2005;280(38):32968-78. DOI:10.1074/jbc.M505515200 |

2. Cockburn, D. and Svensson, B. Surface binding sites in carbohydrate active enzymes: an emerging picture of structural and functional diversity. 2013. In: Lindhorst TK, Rauter AP (eds) SPR carbohydrate chemistry—chemical and biological approaches, vol 39. Royal Society of Chemistry, Cambridge. DOI: 10.1039/9781849737173-00204

3. Cockburn, D., Wilkens, C., Ruzanski, C., Andersen, S., Willum Nielsen, J., Smith, A.M., Field, R.A., Willemoës, M., Abou Hachem, M., and Svensson B. (2014) Analysis of surface binding sites (SBSs) in carbohydrate active enzymes with focus on glycoside hydrolase families 13 and 77 — a mini-review. Biologia, 69, 705-712. DOI: 10.2478/s11756-014-0373-9

4. Cuyvers S, Dornez E, Delcour JA, and Courtin CM. (2012). Occurrence and functional significance of secondary carbohydrate binding sites in glycoside hydrolases. Crit Rev Biotechnol. 2012;32(2):93-107. DOI:10.3109/07388551.2011.561537 |
5. Ludwiczek ML, Heller M, Kantner T, and McIntosh LP. (2007). A secondary xylan-binding site enhances the catalytic activity of a single-domain family 11 glycoside hydrolase. J Mol Biol. 2007;373(2):337-54. DOI:10.1016/j.jmb.2007.07.057 |

6. Gibson, RM, and Svensson, B. Identification of tryptophanyl residues involved in binding of carbohydrate ligands to barley α-amylase 2. Carlsberg Res Commun. 1987. 52: 373-379.

7. Koropatkin NM and Smith TJ. (2010). SusG: a unique cell-membrane-associated alpha-amylase from a prominent human gut symbiont targets complex starch molecules. Structure. 2010;18(2):200-15. DOI:10.1016/j.str.2009.12.010 |
8. Nielsen MM, Bozonnet S, Seo ES, Mótyán JA, Andersen JM, Dilokpimol A, Abou Hachem M, Gyémánt G, Naested H, Kandra L, Sigurskjold BW, and Svensson B. (2009). Two secondary carbohydrate binding sites on the surface of barley alpha-amylase 1 have distinct functions and display synergy in hydrolysis of starch granules. Biochemistry. 2009;48(32):7686-97. DOI:10.1021/bi900795a |
9. Nielsen JW, Kramhøft B, Bozonnet S, Abou Hachem M, Stipp SL, Svensson B, and Willemoës M. (2012). Degradation of the starch components amylopectin and amylose by barley α-amylase 1: role of surface binding site 2. Arch Biochem Biophys. 2012;528(1):1-6. DOI:10.1016/j.abb.2012.08.005 |
10. Fujii K, Minagawa H, Terada Y, Takaha T, Kuriki T, Shimada J, and Kaneko H. (2007). Function of second glucan binding site including tyrosines 54 and 101 in Thermus aquaticus amylomaltase. J Biosci Bioeng. 2007;103(2):167-73. DOI:10.1263/jbb.103.167 |
11. Cuyvers S, Dornez E, Abou Hachem M, Svensson B, Hothorn M, Chory J, Delcour JA, and Courtin CM. (2012). Isothermal titration calorimetry and surface plasmon resonance allow quantifying substrate binding to different binding sites of Bacillus subtilis xylanase. Anal Biochem. 2012;420(1):90-2. DOI:10.1016/j.ab.2011.09.005 |
12. Patrick WM, Nakatani Y, Cutfield SM, Sharpe ML, Ramsay RJ, and Cutfield JF. (2010). Carbohydrate binding sites in Candida albicans exo-β-1,3-glucanase and the role of the Phe-Phe 'clamp' at the active site entrance. FEBS J. 2010;277(21):4549-61. DOI:10.1111/j.1742-4658.2010.07869.x |
13. De Vos D, Collins T, Nerinckx W, Savvides SN, Claeyssens M, Gerday C, Feller G, and Van Beeumen J. (2006). Oligosaccharide binding in family 8 glycosidases: crystal structures of active-site mutants of the beta-1,4-xylanase pXyl from Pseudoaltermonas haloplanktis TAH3a in complex with substrate and product. Biochemistry. 2006;45(15):4797-807. DOI:10.1021/bi052193e |
14. Lo Leggio L, Kalogiannis S, Eckert K, Teixeira SC, Bhat MK, Andrei C, Pickersgill RW, and Larsen S. (2001). Substrate specificity and subsite mobility in T. aurantiacus xylanase 10A. FEBS Lett. 2001;509(2):303-8. DOI:10.1016/s0014-5793(01)03177-5 |
15. Schmidt A, Gübitz GM, and Kratky C. (1999). Xylan binding subsite mapping in the xylanase from Penicillium simplicissimum using xylooligosaccharides as cryo-protectant. Biochemistry. 1999;38(8):2403-12. DOI:10.1021/bi982108l |
16. Vandermarliere E, Bourgois TM, Rombouts S, Van Campenhout S, Volckaert G, Strelkov SV, Delcour JA, Rabijns A, and Courtin CM. (2008). Crystallographic analysis shows substrate binding at the -3 to +1 active-site subsites and at the surface of glycoside hydrolase family 11 endo-1,4-beta-xylanases. Biochem J. 2008;410(1):71-9. DOI:10.1042/BJ20071128 |
17. Mikami B, Adachi M, Kage T, Sarikaya E, Nanmori T, Shinke R, and Utsumi S. (1999). Structure of raw starch-digesting Bacillus cereus beta-amylase complexed with maltose. Biochemistry. 1999;38(22):7050-61. DOI:10.1021/bi9829377 |
18. Sevcík J, Hostinová E, Solovicová A, Gasperík J, Dauter Z, and Wilson KS. (2006). Structure of the complex of a yeast glucoamylase with acarbose reveals the presence of a raw starch binding site on the catalytic domain. FEBS J. 2006;273(10):2161-71. DOI:10.1111/j.1742-4658.2006.05230.x |
19. Allouch J, Helbert W, Henrissat B, and Czjzek M. (2004). Parallel substrate binding sites in a beta-agarase suggest a novel mode of action on double-helical agarose. Structure. 2004;12(4):623-32. DOI:10.1016/j.str.2004.02.020 |
20. Huet J, Rucktooa P, Clantin B, Azarkan M, Looze Y, Villeret V, and Wintjens R. (2008). X-ray structure of papaya chitinase reveals the substrate binding mode of glycosyl hydrolase family 19 chitinases. Biochemistry. 2008;47(32):8283-91. DOI:10.1021/bi800655u |
21. Guce AI, Clark NE, Salgado EN, Ivanen DR, Kulminskaya AA, Brumer H 3rd, and Garman SC. (2010). Catalytic mechanism of human alpha-galactosidase. J Biol Chem. 2010;285(6):3625-3632. DOI:10.1074/jbc.M109.060145 |
22. Varghese JN, Colman PM, van Donkelaar A, Blick TJ, Sahasrabudhe A, and McKimm-Breschkin JL. (1997). Structural evidence for a second sialic acid binding site in avian influenza virus neuraminidases. Proc Natl Acad Sci U S A. 1997;94(22):11808-12. DOI:10.1073/pnas.94.22.11808 |
23. Santos CR, Tonoli CC, Trindade DM, Betzel C, Takata H, Kuriki T, Kanai T, Imanaka T, Arni RK, and Murakami MT. (2011). Structural basis for branching-enzyme activity of glycoside hydrolase family 57: structure and stability studies of a novel branching enzyme from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. Proteins. 2011;79(2):547-57. DOI:10.1002/prot.22902 |
24. Kurakata Y, Uechi A, Yoshida H, Kamitori S, Sakano Y, Nishikawa A, and Tonozuka T. (2008). Structural insights into the substrate specificity and function of Escherichia coli K12 YgjK, a glucosidase belonging to the glycoside hydrolase family 63. J Mol Biol. 2008;381(1):116-28. DOI:10.1016/j.jmb.2008.05.061 |
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