PDBsum entry 2dtm

Immune system

PDB id: 2dtm

Contents

Protein chains

218 a.a.

216 a.a.

Waters ×95

References listed in PDB file

Key reference

Title

Thermodynamic and structural basis for transition-State stabilization in antibody-Catalyzed hydrolysis.

Authors

M.Oda, N.Ito, T.Tsumuraya, K.Suzuki, M.Sakakura, I.Fujii.

Ref.

J Mol Biol, 2007, 369, 198-209. [DOI no: 10.1016/j.jmb.2007.03.023]

PubMed id

17428500

Abstract

Catalytic antibodies 6D9 and 9C10, which were induced by immunization with a haptenic transition-state analog (TSA), catalyze the hydrolysis of a nonbioactive chloramphenicol monoester derivative to generate a bioactive chloramphenicol. These antibodies stabilize the transition state to catalyze the hydrolysis reaction, strictly according to the theoretical relationship: for 6D9, k(cat)/k(uncat)=895 and K(S)/K(TSA)=900, and for 9C10, k(cat)/k(uncat)=56 and K(S)/K(TSA)=60. To elucidate the molecular basis of the antibody-catalyzed reaction, the crystal structure of 6D9 was determined, and the binding thermodynamics of 6D9 and 9C10 with both the substrate and the TSA were analyzed using isothermal titration calorimetry. The crystal structure of the unliganded 6D9 Fab was determined at 2.25 A resolution and compared with that of the TSA-liganded 6D9 Fab reported previously, showing that the TSA is bound into the hydrophobic pocket of the antigen-combining site in an "induced fit" manner, especially at the L1 and H3 CDR loops. Thermodynamic analyses showed that 6D9 binds the substrate of the TSA with a positive DeltaS, differing from general thermodynamic characteristics of antigen-antibody interactions. This positive DeltaS could be due to the hydrophobic interactions between 6D9 and the substrate or the TSA mediated by Trp H100i. The difference in DeltaG between substrate and TSA-binding to 6D9 was larger than that to 9C10, which is in good correlation with the larger k(cat) value of 6D9. Interestingly, the DeltaDeltaG was mainly because of the DeltaDeltaH. The correlation between k(cat) and DeltaDeltaH is suggestive of "enthalpic strain" leading to destabilization of antibody-substrate complexes. Together with X-ray structural analyses, the thermodynamic analyses suggest that upon binding the substrate, the antibody alters the conformation of the ester moiety in the substrate from the planar Z form to a thermodynamically unstable twisted conformation, followed by conversion into the transition state. Enthalpic strain also contributes to the transition-state stabilization by destabilizing the ground state, and its degree is much larger for the more efficient catalytic antibody, 6D9.

Figure 1.
Figure 1. Chemical transformation resulting from antibody-catalyzed prodrug activation and chemical formulae of the compounds used in this study. Antibodies 6D9 and 9C10 were raised against chloramphenicol phosphonate 3 and catalyze the hydrolysis of chloramphenicol ester 1 to generate chloramphenicol 2 and the acid product. Sub-Lys (4) was used for the ITC study.

Figure 4.
Figure 4. Conformational changes of His L27d (a) and Trp H100i (b) induced by TSA binding. The structures of 6D9 in the unliganded form (sky blue) are superimposed on that of the liganded form (blue), form II, re-refined based on the data from the previous work.^10 Binding of the TSA “pulls” the L1 loop through a hydrogen bond between the phosphate and the catalytic His L27d. The indole ring of Trp H100i in the unliganded form occupies a hydrophobic pocket, whereas it flips out to allow the TSA to bind in the pocket in the complex. For clarity, the hapten is represented as the transition-state analog 3.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2007, 369, 198-209) copyright 2007.

Secondary reference #1

Title

A structural basis for transition-State stabilization in antibody-Catalyzed hydrolysis: crystal structures of an abzyme at 1. 8 a resolution.

Authors

O.Kristensen, D.G.Vassylyev, F.Tanaka, K.Morikawa, I.Fujii.

Ref.

J Mol Biol, 1998, 281, 501-511. [DOI no: 10.1006/jmbi.1998.1940]

PubMed id

9698565

Figure 3.
Figure 3. Sketch showing the position of the hapten close to the prominent CDR H3.

Figure 6.
Figure 6. The van der Waals molecular surfaces are found to be highly complementary between the antibody 6D9 combining site (green and cyan) and the hapten (red). Water molecules are shown in yellow. The program XtalView (McRee, 1992) was used to generate the surfaces and provided an interface to render the Figure with Raster3D (Meritt & Murphy, 1994).

The above figures are reproduced from the cited reference with permission from Elsevier