A Mutational Analysis of Binding Interactions in an Antigen-Antibody Protein-Protein Complex

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• 作者：William Dall'Acqua ; Ellen R. Goldman ; Wenhong Lin ; Connie Teng ; Daisuke Tsuchiya ; Hongmin Li ; Xavier Ysern ; Bradford C. Braden ; Yili Li ; Sandra J. Smith-Gill ; and Roy A. Mariuzza
• 刊名：Biochemistry
• 出版年：1998
• 出版时间：June 2, 1998
• 年：1998
• 卷：37
• 期：22
• 页码：7981 - 7991
• 全文大小：130K
• 年卷期：v.37,no.22(June 2, 1998)
• ISSN：1520-4995

文摘

Alanine scanning mutagenesis, double mutant cycles, andX-ray crystallography were used tocharacterize the interface between the anti-hen egg white lysozyme(HEL) antibody D1.3 and HEL. Twelveout of the 13 nonglycine contact residues on HEL, as determined by thehigh-resolution crystal structureof the D1.3-HEL complex, were individually truncated to alanine.Only four positions showed a ges/gifchars/Delta.gif" BORDER=0 >ges/gifchars/Delta.gif" BORDER=0 >G(ges/gifchars/Delta.gif" BORDER=0 >G_mutant -ges/gifchars/Delta.gif" BORDER=0 >G_wild-type) of greater than 1.0 kcal/mol,with HEL residue Gln121 proving the most criticalfor binding (ges/gifchars/Delta.gif" BORDER=0 >ges/gifchars/Delta.gif" BORDER=0 >G = 2.9 kcal/mol). These residuesform a contiguous patch at the periphery of the epitoperecognized by D1.3. To understand how potentially disruptivemutations in the antigen are accommodatedin the D1.3-HEL interface, we determined the crystal structure to 1.5Å resolution of the complex betweenD1.3 and HEL mutant Asp18 ges/entities/rarr.gif"> Ala. This mutation results in ages/gifchars/Delta.gif" BORDER=0 >ges/gifchars/Delta.gif" BORDER=0 >G of only 0.3 kcal/mol, despite theloss of a hydrogen bond and seven van der Waals contacts to the Asp18side chain. The crystal structurereveals that three additional water molecules are stably incorporatedin the antigen-antibody interface atthe site of the mutation. These waters help fill the cavitycreated by the mutation and form part of arearranged solvent network linking the two proteins. To furtherdissect the energetics of specific interactionsin the D1.3-HEL interface, double mutant cycles were carried out tomeasure the coupling of 14 aminoacid pairs, 10 of which are in direct contact in the crystal structure.The highest coupling energies, 2.7and 2.0 kcal/mol, were measured between HEL residue Gln121 and D1.3residues V_LTrp92 and V_LTyr32, respectively. The interaction between Gln121 andV_LTrp92 consists of three van der Waalscontacts,while the interaction of Gln121 with V_LTyr32 ismediated by a hydrogen bond. Surprisingly, however,most cycles between interface residues in direct contact in the crystalstructure showed no significantcoupling. In particular, a number of hydrogen-bonded residue pairswere found to make no net contributionto complex stabilization. We attribute these results toaccessibility of the mutation sites to water, suchthat the mutated residues exchange their interaction with each other tointeract with water. This impliesthat the strength of the protein-protein hydrogen bonds in theseparticular cases is comparable to that ofthe protein-water hydrogen bonds they replace. Thus, the simplefact that two residues are in directcontact in a protein-protein interface cannot be taken as evidencethat there necessarily exists a productiveinteraction between them. Rather, the majority of such contactsmay be energetically neutral, as in theD1.3-HEL complex.