Ligand binding preferences probed by ESI MS and amide H/D exchange

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1 Ligand binding preferences probed by ESI MS and amide H/D exchange Hui Xiao; Benjamin W. Benson; Stephen J. Eyles; Igor A. Kaltashov Department of Chemistry, University of Massachusetts, Amherst, MA 01003

2 VERVIEW PURPSE Investigate conformational dynamics and ligand-binding mechanisms of cellular retinoic acid binding protein (CRABP I) Determine binding preferences of this protein using all-trans retinoic acid (RA), 9-cis RA, 13-cis RA and retinol (RH) METHD Electrospray mass spectrometry Amide hydrogen/deuterium exchange RESULTS H/D exchange can be used to assess ligand binding specificity. Diminishing affinity of CRABP I to RA isomers is in the following order: all trans>13-cis>9-cis and no binding to RH.

3 INTRDUCTIN All-trans retinoic (RA) acid is a major physiologically active metabolite of vitamin A. It is readily degraded in vitro to produce stereoisomers (mainly 9-cis and 13-cis), as well as its own oxidized forms. Water-insoluble RA is sequestered and transported in cytosole by a 16 kda protein, cellular retinoic acid binding protein I (CRABP I). Although several isomers of RA have been also detected in vivo, their carriers have not been identified 1. We have been investigating conformational dynamics and RAbinding mechanisms of CRABP I using ESI MS and amide H/D exchange 2,3. We are now extending this approach to study conformational preferences of CRABP I using RA, 9-cis RA, 13-cis RA and retinol. We are also investigating a possible role of CRABP I as a protector of RA against photo-isomerization.

4 2 C(CH 2 ) 7 CH 3 H Retinol(Viatamin A) Retinyl Palmitate H Retinal β-carotene H All-trans Retinoic acid (RA) 11-cis-Retinal H 9-cis RA H 13-cis RA H 9-13-di-cis RA H H H H 4-H-RA 4-oxo-RA Metablites and derivatives of retinol (vitamin A) and retinoic acid. Adapted from Napoli, J. L., Biochim. Biophys. Acta, 1999, 1440, 139

5 MATERIALS & METHDS Instrumentation JMS-700 MStation (JEL, Tokyo, Japan) two-sector mass spectrometer Esquire-LC electrospray ion trap mass spectrometer (Bruker Daltonics, Billerica, MA, USA). Sample preparation Deuterated protein was prepared by replacing all the labile hydrogen atoms with deuterium in D 2 and CD 3 C 2 D buffer solution, followed by lyophilization. A lyophilized protein sample was dissolved to a concentration of ~1 mg/ml with D 2 and CD 3 C 2 D buffer solution. Back-exchange was initiated by diluting the protein solution 1:50 in a desired ph level buffer solution.

6 ESI MS data indicate that all-trans RA has higher affinity than 9-cis- RA and 13 cis-ra. The order of these three isomers binding affinity with CRABP is all-trans RA>>13-cis-RA>9-cis-RA with no binding to RH as judged by relative intensities of holo- and apo-protein peaks in the spectra of CRABP/retinoid mixtures acquired at neutral ph. However, gas phase interactions do not necessarily reflect the protein ligand interactions in solution, particularly if hydrophobic forces play an important role. In the lock-and-key type of interaction stronger binding is expected to induce higher amide protection. The ligand binding results in significant decrease of the free energy of the native conformation, thus affecting the equilibrium between this state and the transiently populated intermediate states. This shift of equilibrium is reflected in a significantly increased amide hydrogen protection of the protein backbone in the presence of the ligand. Therefore, we carried out H/D exchange experiment to verify the binding preferences of RA isomers and RH.

7 H/D exchange kinetics of CRABP I in the presence of RA, 13-cis-RA, 9-cis-RA, RH 90 number of protected amides m M N H 4 CH 3 C 2 all-trans 13-cis 9-cis re tinol apo tim e (m inutes)

8 ligand binding A schematic representation of a energy landscapes for the protein-small ligand interaction ( lock-and-key model). Vertical dimension represents free energy, and horizontal dimension represents conformational entropy. RA, 9-cis-RA, 13-cis-RA binding with protein results in higher amide protection as compared with apo-protein, while RH does not affect the backbone protection.

9 All-trans RA undergoes photo-isomerization. We have investigated possible protective effect of CRABP I on RA. Isomerization was monitored by HPLC and HPLC-ESI/MS. H hν + etc H Protein hν H?

10 λ=340 nm (arbitrary units) All-trans RA All-trans RA after 2 hour exposure to UV light (λ=365 nm) All-trans RA after 2 hour exposure to UV light (λ=365 nm) in the presence of CRABP I (2:1 molar excess) All-trans RA after 2 hour exposure to UV light (λ=365 nm) in the presence of CRABP I (5:1 molar excess) *# # *# Retention time (min) Photo-isomerization rate of RA( λ=340 nm) is reduced in the presence of CRABP I. Isomers of RA were identified using commercially available standards (*) and /or HPLC/ESI/MS (#).

11 100 HPLC-MS chromatograms of All-trans RA after 2 hour exposure to UV light (λ=365 nm) (BPC) Peak 1, min Peak 2, min Peak 3, min Peak 4, min Arbitrary units Retention time (min) 25.0

12 Identification of photo-isomers of RA by HPLC-MS Peak 1, min Peak 2, min m/z m/z Peak 3, min Peak 4, min m/z m/z

13 CNCLUSINS Ligand binding to protein results in significant changes in conformational stability. CRABP I binding to RA isomers can be assessed independently by (i) comparing the relative intensities of the holo-and apo-protein peaks in the ESI spectra of the protein/retinoid mixtures and (ii) measuring H/D exchange rates of holo- and apo-protein. CRABP I binds RA isomers in the following order: all-trans>>13-cis- RA 9-cis-RA>> RH (no binding). All-trans RA bound to CRABP I has some (but not full) protection from photo-isomerization, indicating highly dynamic nature of the protein-ligand complex.

14 FUTURE DIRECTINS Using ESI/CAD in combination with H/D exchange to determine the local conformational information. Study the H/D exchange under EX1 conditions( by employing mildly denaturing condition); detect partially unfolded states essential for ligand binding and probe the protein dynamics. Extend the study to site-directed mutants of CRABP I to explore the ligand binding site.

15 REFERENCE 1. Napoli, J. L., Biochim. Biophys. Acta, 1999, 1440, Eyles, S. J., Dresch, T., Gierasch, L.M. and Kaltashov, I. A., J. Mass Spectrom., 1999, 34, Eyles, S. J., Speir, J. P., Kruppa, G. H., Gierasch, L.M. and Kaltashov, I. A., J. Am. Chem. Soc., 2000, 122, 495. ACKNWLEDGEMENTS NIH R01GM61666 ASMS Young Investigator Award Mass Spectrometry Center at University of Massachusetts