AutoLink: Automated sequential resonance assignment of biopolymers from NMR data by relative-hypothesis-prioritization-based simulated logic

The receptor for advanced glycated end products (RAGE) is a multiligand receptor that is implicated in the pathogenesis of various diseases, including diabetic complications, neurodegenerative disorders, and inflammatory responses. The ability of RAGE to recognize advanced glycated end products (AGEs) formed by nonenzymatic glycoxidation of cellular proteins places RAGE in the category of pattern recognition receptors. The structural mechanism of AGE recognition was an enigma due to the diversity of chemical structures found in AGE-modified proteins. Here, using NMR spectroscopy we showed that the immunoglobulin V-type domain of RAGE is responsible for recognizing various classes of AGEs. Three distinct surfaces of the V domain were identified to mediate AGE-V domain interactions. They are located in the positively charged areas of the V domain. The first interaction surface consists of strand C and loop CC, the second interaction surface consists of strand C, strand F, and loop FG, and the third interaction surface consists of strand A and loop EF. The secondary structure elements of the interaction surfaces exhibit significant flexibility on the ms-s time scale. Despite highly specific AGE-V domain interactions, the binding affinity of AGEs for an isolated V domain is low, ϳ10 M. Using in-cell fluorescence resonance energy transfer we show that RAGE is a constitutive oligomer on the plasma membrane. We propose that constitutive oligomerization of RAGE is responsible for recognizing patterns of AGE-modified proteins with affinities less than 100 nM.

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“…All spectra were processed using TOPSPIN 2.1 (Bruker, Inc), and assignments were made using CARA (36).…”

Section: Methodsmentioning

confidence: 99%

Structural Basis for Pattern Recognition by the Receptor for Advanced Glycation End Products (RAGE)

Xie

Reverdatto

Frolov

et al. 2008

Journal of Biological Chemistry

219

245

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“…NMR data were processed and analyzed using the nmrPipe and CARA (22,23). To color significance of chemical shift changes on the CH2 backbone structure, a normalized chemical shift changes (Equation 1),…”

Section: H-mentioning

confidence: 99%

Engineered Human Antibody Constant Domains with Increased Stability

Gong¹,

Vu²,

Yang³

et al. 2009

Journal of Biological Chemistry

111

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The immunoglobulin (Ig) constant CH2 domain is critical for antibody effector functions. Isolated CH2 domains are promising as scaffolds for construction of libraries containing diverse binders that could also confer some effector functions. However, previous work has shown that an isolated murine CH2 domain is relatively unstable to thermally induced unfolding. To explore unfolding mechanisms of isolated human CH2 and increase its stability ␥1 CH2 was cloned and a panel of cysteine mutants was constructed. Human ␥1 CH2 unfolded at a higher temperature (T m ‫؍‬ 54.1°C, as measured by circular dichroism) than that previously reported for a mouse CH2 (41°C). One mutant (m01) was remarkably stable (T m ‫؍‬ 73.8°C). Similar results were obtained by differential scanning calorimetry. This mutant was also significantly more stable than the wild-type CH2 against urea induced unfolding (50% unfolding at urea concentration of 6.8 M versus 4.2 M). The m01 was highly soluble and monomeric. The existence of the second disulfide bond in m01 and its correct position were demonstrated by mass spectrometry and nuclear magnetic resonance spectroscopy, respectively. The loops were on average more flexible than the framework in both CH2 and m01, and the overall secondary structure was not affected by the additional disulfide bond. These data suggest that a human CH2 domain is relatively stable to unfolding at physiological temperature, and that both CH2 and the highly stable mutant m01 are promising new scaffolds for the development of therapeutics against human diseases. Monoclonal antibodies (mAbs)2 with high affinity and specificity are now well established therapeutics and invaluable tools for biological research. It appears that their use will continue to expand in both targets and disease indications. However, a fundamental problem for full-size mAbs that limits their applications is their poor penetration into tissues (e.g. solid tumors) and poor or absent binding to regions on the surface of some molecules (e.g. on the HIV envelope glycoprotein) that are accessible by molecules of smaller size. Antibody fragments, e.g. Fabs (ϳ60 kDa) or single chain Fv fragments (scFvs) (20ϳ30 kDa), are significantly smaller than full-size antibodies (ϳ150 kDa), and have been used as imaging reagents and candidate therapeutics. Even smaller fragments of antibodies are of great interest and advantageous for pharmaceutical applications including cancer targeting and imaging.During the last decade a large amount of work has been aimed at developing of small size binders with scaffolds based on various highly stable human and non-human molecules (1-8). A promising direction is the development of binders based on the heavy or light chain variable region of an antibody; these fragments ranging in size from 11 kDa to 15 kDa were called "domain antibodies" or "dAbs" (7, 9). A unique kind of antibodies composed only of heavy chains are naturally formed in camels, dromedaries, and llamas, and their variable regions can also recognize antigens as sin...

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“…To obtain steady state 15 N nuclear Overhauser effect (NOE) values for ctRAGE, standard experiments were used (23). All spectra were processed using TOPSPIN 2.1 (Bruker, Inc.), and assignments were made by using CARA (24).…”

mentioning

confidence: 99%

Signal Transduction in Receptor for Advanced Glycation End Products (RAGE)

Rai

Maldonado

Burz

et al. 2012

Journal of Biological Chemistry

101

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Background: RAGE is implicated in diabetes complications, inflammation, and neurodegeneration. Results: Cytosolic domain of RAGE, ctRAGE, contains an unusual ␣-turn that mediates the mDia1-ctRAGE interaction and is required for RAGE-dependent signaling. Conclusion: A novel mechanism through which extracellular RAGE ligands regulate RAGE-mDia1 signaling is established. Significance: A novel binding interface as a target for suppression of RAGE ligand-stimulated signal transduction is identified.

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AutoLink: Automated sequential resonance assignment of biopolymers from NMR data by relative-hypothesis-prioritization-based simulated logic

Cited by 81 publications

References 35 publications

Structural Basis for Pattern Recognition by the Receptor for Advanced Glycation End Products (RAGE)

Structural Basis for Pattern Recognition by the Receptor for Advanced Glycation End Products (RAGE)

Engineered Human Antibody Constant Domains with Increased Stability

Signal Transduction in Receptor for Advanced Glycation End Products (RAGE)

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