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 (named SBS1 and SBS2) 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 Ludwiczek2007 Cuyvers2012b. 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]).

References

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 |
11. 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 |
12. 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 |
13. 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 |