Anchor residues in protein–protein interactions

Rajamani, Deepa; Thiel, Spencer; Vajda, Sándor; Camacho, Carlos J.

doi:10.1073/pnas.0401942101

Cited by 319 publications

(383 citation statements)

References 27 publications

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“…The IC 50 values for all of the other variants of RNase 1 listed in Table 3 fall outside the measurable range of the assay (IC 50 > 25 μM). R39L/N67L/N88A/G89L/R91L RNase 1 and N67D/N88A/G89D/R91D RNase 1 killed approximately 60% of the K-562 cells at 25 μM (Figure 6b), indicative of IC 50 values only slightly above 25 μM.…”

Section: Molecular Chargementioning

confidence: 94%

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Inhibition of Human Pancreatic Ribonuclease by the Human Ribonuclease Inhibitor Protein

Johnson

McCoy

Bingman

et al. 2007

Journal of Molecular Biology

135

201

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The ribonuclease inhibitor protein (RI) binds to members of the bovine pancreatic ribonuclease (RNase A) superfamily with an affinity in the femtomolar range. Here, we report on structural and energetic aspects of the interaction between human RI (hRI) and human pancreatic ribonuclease (RNase 1). The structure of the crystalline hRI·RNase 1 complex was determined at a resolution of 1.95 Å, revealing the formation of 19 intermolecular hydrogen bonds involving 13 residues of RNase 1. In contrast, only 9 such hydrogen bonds are apparent in the structure of the complex between porcine RI and RNase A. hRI, which is anionic, also appears to use its horseshoe-shaped structure to engender long-range Coulombic interactions with RNase 1, which is cationic. In accordance with the structural data, the hRI·RNase 1 complex was found to be extremely stable (t ½ = 81 days; K d = 2.9 × 10 -16 M). Site-directed mutagenesis experiments enabled the identification of two cationic residues in RNase 1-Arg39 and Arg91-that are especially important for both the formation and stability of the complex, and are thus termed "electrostatic targeting residues". Disturbing the electrostatic attraction between hRI and RNase 1 yielded a variant of RNase 1 that maintained ribonucleolytic activity and conformational stability but had a 2.8 × 10 3 -fold lower association rate for complex formation and 5.9 × 10 9 -fold lower affinity for hRI. This variant of RNase 1, which exhibits the largest decrease in RI affinity of any engineered ribonuclease, is also toxic to human erythroleukemia cells. Together, these results provide new insight into an unusual and important protein-protein interaction, and could expedite the development of human ribonucleases as chemotherapeutic agents.

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Section: Molecular Chargementioning

confidence: 94%

“…50 were calculated by fitting the curves using nonlinear regression to equation (4), wherein y is the total DNA synthesis following the [methyl-3 H]thymidine pulse, and h is the slope of the curve. Electron density at 1σ (2F obs -F calc ) of key contact residues between hRI (green) and RNase 1 (purple).…”

Section: Cytotoxicitymentioning

confidence: 99%

Inhibition of Human Pancreatic Ribonuclease by the Human Ribonuclease Inhibitor Protein

Johnson

McCoy

Bingman

et al. 2007

Journal of Molecular Biology

135

201

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“…The Thr-47 of HEWL (Thr-47 L ) fits into this cavity and is surrounded by residues occluding solvent from this site (Fig. 1C), suggesting that Thr-47 L is an energetic hot spot of the VHH-HEWL interaction (32,34). Indeed, Thr-47 L has in both cases the highest ⌬ASA of all epitope residues, accounting for 18 and 20%, and making two hydrogen bonds with cAb-Lys2 and three with D2-L19.…”

Section: Ontogeny Of D2-l19 and Cab-lys2-twomentioning

confidence: 99%

Strong in Vivo Maturation Compensates for Structurally Restricted H3 Loops in Antibody Repertoires

Genst

Silence

Ghahroudi

et al. 2005

Journal of Biological Chemistry

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A central paradigm in immunology states that successful generation of high affinity antibodies necessitates an immense primary repertoire of antigen-combining sites. Much of the diversity of this repertoire is provided by varying one antigen binding loop, created by inserting randomly a D (diversity) gene out of a small pool between the V and J genes. It is therefore assumed that any particular D-encoded region surrounded by different V and J regions adopts a different conformation. We have solved the structure of two lysozyme-specific variable domains of heavy-chain antibodies isolated from two strictly unrelated dromedaries. These antibodies recombined identical D gene sequences to different V and J precursors with significant variance in their V(D)J junctions. Despite these large differences, the D-encoded loop segments adopt remarkably identical architectures, thus directing the antibodies toward identical epitopes. Furthermore, a striking convergent maturation process occurred in the V region, adapting both binders for their sub-nanomolar affinity association with lysozyme. Hence, on a structural level, humoral immunity may rely more on well developed maturation and selection systems than on the acquisition of large primary repertoires.The interaction of conventional antibodies with antigens is mediated by up to six dedicated hypervariable loops, three (H1-H3) 1 in the variable domain of the heavy chain (VH) and three (L1-L3) in the variable domain of the light chain (VL) (1). Within the antigen-combining site, the H3 loop is the major determinant of antibody diversity and the major contributor for overall antigen affinity and specificity (1-3). This loop is generated after recombining one V, one D, and one J gene out of a pool. Imprecision in the V(D)J recombination event, with concomitant nucleotide deletions and N or P nucleotide additions (4, 5), makes it possible for a particular D gene to occur in different locations and reading frames within the H3-encoded region (4, 5). Hence, the sequence of the H3 loop becomes the most diverse of all antigen binding loops.The crystal structure determination of different antibodies revealed the presence of canonical structures for all antigen binding loops (L1-L3, H1, and H2) except for the H3 loop (6, 7). The folding of a particular loop into a canonical structure is dictated by a small number of conserved key residues within the hypervariable sequences. The immense sequence diversity of the H3 loop with the D-encoded part, which can be located anywhere within the loop, obviously precludes an easy assignment of such key residues. Moreover, because the number of possible V(D)J recombinations exceeds by far the number of crystal structures that are available, it is as yet impossible to investigate whether the D-encoded part of the loop adopts different or similar backbone architectures when present in a different V-J surrounding. Likewise, it remains an open question whether the same epitope on a large antigen will be recognized by antibodies originating from differ...

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“…Evolutionarily conserved anchor residues provide specific side chains that penetrate into a structurally constrained binding groove of the binding partner during molecular recognition between two interacting proteins (32). Such recognition motifs bury the maximum surface area after complexation.…”

Section: Discussionmentioning

confidence: 99%

Interaction of CheY with the C-Terminal Peptide of CheZ

Guhaniyogi¹,

Patel³

et al. 2008

J Bacteriol

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Chemotaxis, a means for motile bacteria to sense the environment and achieve directed swimming, is controlled by flagellar rotation. The primary output of the chemotaxis machinery is the phosphorylated form of the response regulator CheY ( In this study, we report biochemical and structural data that support the alternate-binding-mode hypothesis and identify key recognition elements in the CheY-CheZ C interaction. In addition, we present kinetic studies of the CheZ C -associated effect on CheY phosphorylation with its physiologically relevant phosphodonor, the histidine kinase CheA. Our results indicate mechanistic differences in phosphotransfer from the kinase CheA versus that from small-molecule phosphodonors, explaining a modest twofold increase of CheY phosphorylation with the former, observed in this study, relative to a 10-fold increase previously documented with the latter.

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Anchor residues in protein–protein interactions

Cited by 319 publications

References 27 publications

Inhibition of Human Pancreatic Ribonuclease by the Human Ribonuclease Inhibitor Protein

Inhibition of Human Pancreatic Ribonuclease by the Human Ribonuclease Inhibitor Protein

Strong in Vivo Maturation Compensates for Structurally Restricted H3 Loops in Antibody Repertoires

Interaction of CheY with the C-Terminal Peptide of CheZ

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