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Infection and Immunity, August 2002, p. 4485-4493, Vol. 70, No. 8
0019-9567/02/$04.00+0     DOI: 10.1128/IAI.70.8.4485-4493.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Structure-Function Analysis of Decay-Accelerating Factor: Identification of Residues Important for Binding of the Escherichia coli Dr Adhesin and Complement Regulation

Rafia J. Hasan,1,2 Edyta Pawelczyk,2 Petri T. Urvil,1 Mathura S. Venkatarajan,3 Pawel Goluszko,1 Jozef Kur,4 Rangaraj Selvarangan,2,{dagger} Stella Nowicki,1,2 Werner A. Braun,3 and Bogdan J. Nowicki1,2*

Departments of Obstetrics & Gynecology,1 Microbiology & Immunology,2 Human Biological Chemistry & Genetics, The University of Texas Medical Branch, Galveston, Texas,3 Technical University of Gdansk, 80-952 Gdansk, Poland4

Received 7 January 2002/ Returned for modification 20 February 2002/ Accepted 13 May 2002

Decay-accelerating factor (DAF), a complement regulatory protein, also serves as a receptor for Dr adhesin-bearing Escherichia coli. The repeat three of DAF was shown to be important in Dr adhesin binding and complement regulation. However, Dr adhesins do not bind to red blood cells with the rare polymorphism of DAF, designated Dr(a-); these cells contain a point mutation (Ser165-Leu) in DAF repeat three. In addition, monoclonal antibody IH4 specific against repeat three was shown to block both Dr adhesin binding and complement regulatory functions of DAF. Therefore, to identify residues important in binding of Dr adhesin and IH4 and in regulating complement, we mutated 11 amino acids—predominantly those in close proximity to Ser165 to alanine—and expressed these mutations in Chinese hamster ovary cells. To map the mutations, we built a homology model of repeat three based on the poxvirus complement inhibitory protein, using the EXDIS, DIAMOD, and FANTOM programs. We show that perhaps Ser155, and not Ser165, is the key amino acid that interacts with the Dr adhesin and amino acids Gly159, Tyr160, and Leu162 and also aids in binding Dr adhesin. The IH4 binding epitope contains residues Phe148, Ser155, and L171. Residues Phe123 and Phe148 at the interface of repeat 2-3, and also Phe154 in the repeat three cavity, were important for complement regulation. Our results show that residues affecting the tested functions are located on the same loop (148 to 171), at the same surface of repeat three, and that the Dr adhesin-binding and complement regulatory epitopes of DAF appear to be distinct and are {approx}20 Å apart.

* Corresponding author. Mailing address: 301 University Blvd., Route 1062, Galveston, TX 77555-1062. Phone: (409) 772-7599. Fax: (409) 747-0475. E-mail: bnowicki{at}utmb.edu.

Editor: V. J. DiRita

{dagger} Present address: Department of Laboratory Medicine, Division of Clinical Microbiology, University of Washington, Seattle, WA 98195-7110.

Infection and Immunity, August 2002, p. 4485-4493, Vol. 70, No. 8
0019-9567/02/$04.00+0     DOI: 10.1128/IAI.70.8.4485-4493.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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