PDBsum entry: 1kxq

Hydrolase, immune system

PDB id: 1kxq

Contents

Protein chains

496 a.a. *

120 a.a. *

Metals

_CL ×4

_CA ×4

Waters ×2807

* Residue conservation analysis

PDB id:

1kxq

Name:

Hydrolase, immune system

Title:

Camelid vhh domain in complex with porcine pancreatic alpha- amylase

Structure:

Alpha-amylase, pancreatic. Chain: a, b, c, d. Synonym: 1,4-alpha-d-glucan glucanohydrolase. Antibody vhh fragment cabamd9. Chain: e, f, g, h. Engineered: yes

Source:

Sus scrofa. Pig. Organism_taxid: 9823. Other_details: pancreatic enzyme. Camelus dromedarius. Arabian camel. Organism_taxid: 9838. Expressed in: escherichia coli. Expression_system_taxid: 562.

Biol. unit:

Dimer (from PQS)

Resolution:

1.60Å R-factor: 0.197 R-free: 0.219

Authors:

A.Desmyter,S.Spinelli,F.Payan,M.Lauwereys,L.Wyns, S.Muyldermans,C.Cambillau

Key ref:

A.Desmyter et al. (2002). Three camelid VHH domains in complex with porcine pancreatic alpha-amylase. Inhibition and versatility of binding topology. J Biol Chem, 277, 23645-23650. PubMed id: 11960990 DOI: 10.1074/jbc.M202327200

Date:

01-Feb-02 Release date: 19-Jun-02

Protein chains

P00690  (AMYP_PIG) -  Pancreatic alpha-amylase

Seq: 511 a.a.

Struc: 496 a.a.*

Protein chain

	No UniProt id for this chain
	* PDB and UniProt seqs differ at 7 residue positions (black crosses)

Enzyme reactions

• Enzyme class: Chains A, B, C, D: E.C.3.2.1.1 - Alpha-amylase.
• Reaction: Endohydrolysis of 1,4-alpha-glucosidic linkages in oligosaccharides and polysaccharides.

 Gene Ontology (GO) functional annotation 

• Cellular component: extracellular region (2 terms)
• Biological process: metabolic process (3 terms)
• Biochemical function: catalytic activity (8 terms)

DOI no: 10.1074/jbc.M202327200

J Biol Chem 277:23645-23650 (2002)

PubMed id: 11960990

Three camelid VHH domains in complex with porcine pancreatic alpha-amylase. Inhibition and versatility of binding topology.

A.Desmyter, S.Spinelli, F.Payan, M.Lauwereys, L.Wyns, S.Muyldermans, C.Cambillau.

ABSTRACT

Camelids produce functional antibodies devoid of light chains and CH1 domains. The antigen-binding fragment of such heavy chain antibodies is therefore comprised in one single domain, the camelid heavy chain antibody VH (VHH). Here we report on the structures of three dromedary VHH domains in complex with porcine pancreatic alpha-amylase. Two VHHs bound outside the catalytic site and did not inhibit or inhibited only partially the amylase activity. The third one, AMD9, interacted with the active site crevice and was a strong amylase inhibitor (K(i) = 10 nm). In contrast with complexes of other proteinaceous amylase inhibitors, amylase kept its native structure. The water-accessible surface areas of VHHs covered by amylase ranged between 850 and 1150 A(2), values similar to or even larger than those observed in the complexes between proteins and classical antibodies. These values could certainly be reached because a surprisingly high extent of framework residues are involved in the interactions of VHHs with amylase. The framework residues that participate in the antigen recognition represented 25-40% of the buried surface. The inhibitory interaction of AMD9 involved mainly its complementarity-determining region (CDR) 2 loop, whereas the CDR3 loop was small and certainly did not protrude as it does in cAb-Lys3, a VHH-inhibiting lysozyme. AMD9 inhibited amylase, although it was outside the direct reach of the catalytic residues; therefore it is to be expected that inhibiting VHHs might also be elicited against proteases. These results illustrate the versatility and efficiency of VHH domains as protein binders and enzyme inhibitors and are arguments in favor of their use as drugs against diabetes.

Selected figure(s)

Figure 1.
Fig. 1. Sequences and three-dimensional structures of the three VHHs. The three CDRs (1-3) are colored red, green, and blue, respectively, and the cysteines are purple. A, anti-PPA AMB7, AMD9, and AMD10 VHH amino acid sequences. The numbering is according to Kabat ( 30). B, from left to right, views of the AMB7, AMD9, and AMD10 VHHs in the same orientation with the CDR3 oriented in front (view made with SPOCK (33)).

Figure 2.
Fig. 2. Views of the three VHHs superimposed on PPA with the acarbose molecule bound (pink). The PPA surface is in gray, and its visible catalytic residue Asp-300 is in red. The C trace of the VHHs is orange, the three CDRs (1-3) are colored red, green, and blue, respectively, and each VHH surface is transparent. Inset, close-up view of the PPA active site with the inhibitory AMD9 VHH bound. The saccharidic inhibitor acarbose has been positioned in the active site according to the x-ray structure as a probe of the saccharide position. Two residues of the VHH, Tyr-52 and Arg-52a (CPK yellow and orange), clash with the modeled acarbose (views made with SPOCK (33)).

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2002, 277, 23645-23650) copyright 2002.