The Signature 3-O-Sulfo Group of the Anticoagulant Heparin Sequence Is Critical for Heparin Binding to Antithrombin but Is Not Required for Allosteric Activation

Richard, Benjamin; Swanson, Richard; Olson, Steven T.

doi:10.1074/jbc.m109.029892

Cited by 42 publications

(34 citation statements)

References 46 publications

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“…The drift time measured for hexasaccharide-bound ATIII was the same as that measured for Arixtra-bound ATIII, which agrees with a previous study that concluded that removing the 3- O sulfo group affected the equilibrium of native ATIII and activated ATIII but not the conformational change associated with the equilibrium. 53 In other words, this control Hp oligosaccharide retains the specificity of binding, but has a reduced binding affinity.…”

Section: Resultsmentioning

confidence: 99%

Investigating changes in the gas-phase conformation of Antithrombin III upon binding of Arixtra using traveling wave ion mobility spectrometry (TWIMS)

Zhao

Singh

et al. 2015

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We validate the utility of ion mobility to measure protein conformational changes induced by the binding of glycosaminoglycan ligands, using the well characterized system of Antithrombin III (ATIII) and Arixtra, a pharmaceutical agent with heparin (Hp) activity. Heparin has been used as a therapeutic anticoagulant drug for several decades through its interaction with ATIII, a serine protease inhibitor that plays a central role in the blood coagulation cascade. This interaction induces conformational changes within ATIII that dramatically enhance the ATIII-mediated inhibition rate. Arixtra is the smallest synthetic Hp containing the specific pentasaccharide sequence required to bind with ATIII. Here we report the first travelling wave ion mobility mass spectrometry (TWIMS) investigation of the conformational changes in ATIII induced by its interaction with Arixtra. Native electrospray ionization mass spectrometry allowed the gentle transfer of the native topology of ATIII and ATIII–Arixtra complex. IM measurements of ATIII and ATIII–Arixtra complex showed a single structure, with well-defined collisional cross section (CCS) values. An average 3.6% increase in CCS of ATIII occurred as a result of its interaction with Arixtra, which agrees closely with the theoretical estimation of the change in CCS based on protein crystal structures. A comparison of the binding behavior of ATIII under both denaturing and non-denaturing conditions confirmed the significance of a folded tertiary structure of ATIII for its biological activity. A Hp oligosaccharide whose structure is similar to Arixtra but missing the 3-O sulfo group on the central glucosamine residue showed a dramatic decrease in binding affinity towards ATIII, but no change in the mobility behavior of the complex, consistent with prior studies that suggested that 3-O sulfation affects the equilibrium constant for binding to ATIII, but not the mode of interaction. In contrast, nonspecific binding by a Hp tetrasaccharide showed more complex mobility behavior, suggesting more promiscuous interactions with ATIII. The effect of collisional activation of ATIII and ATIII–Arixtra complex were also assessed, revealing that the binding of Arixtra provided ATIII with additional stability against unfolding. Overall, our results validate the capability of TWIMS to retain the significant features of the solution structure of a protein–carbohydrate complex so that it can be used to study protein conformational changes induced by the binding of glycosaminoglycan ligands.

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Section: Resultsmentioning

confidence: 99%

Investigating changes in the gas-phase conformation of Antithrombin III upon binding of Arixtra using traveling wave ion mobility spectrometry (TWIMS)

Zhao

Singh

et al. 2015

Analyst

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“…These features are thought to represent the basis for the ability of HS chains to strongly and specifically interact with proteins (23,25,48). Some proteins such as fibroblast growth factor 2 (FGF2) and antithrombin require specific modifications of HS for optimal binding including 3-O sulfation (49)(50)(51), whereas other proteins such as IL-8 and thrombin mainly rely on HS domain structure or charge density, respectively (52,53). At present, relatively little is known about HS structural features mediating BMP interactions and whether different BMPs require distinct HS modifications and segments for optimal binding (48,54), though the BMP antagonist Noggin preferentially binds to HS carrying N-, 6-O-and 2-O-sulfates (55).…”

Section: Discussionmentioning

confidence: 99%

Domains with highest heparan sulfate–binding affinity reside at opposite ends in BMP2/4 versus BMP5/6/7: Implications for function

Billings

Yang

Mundy

et al. 2018

Journal of Biological Chemistry

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Signaling proteins, including bone morphogenetic proteins (BMPs), specifically interact with heparan sulfate (HS). These interactions regulate protein distribution and function and are largely mediated by domains rich in basic amino acids. The N-terminal region of BMP2 and BMP4 contains one such domain with a typical Cardin-Weintraub (CW) motif, but it is unclear whether the same occurs in BMP5, BMP6 and BMP7 that constitute a separate evolutionary subgroup. Peptides spanning the N-terminal domain of BMP2/4 interacted with substrate-bound HS with nanomolar affinity, but peptides spanning BMP5/6/7 N-terminal domain did not. We reexamined the entire BMP5/6/7 sequences and identified a novel CW-like motif at their Cterminus. Peptides spanning this domain displayed high affinity HS binding, but corresponding BMP2/4 C-terminal peptides did not, likely because of acidic or non-charged residue substitutions. Peptides pre-assembled into NeutrAvidin tetramers displayed the same exact binding selectivity of respective monomers, but bound HS with greater affinity. Tests of possible peptide biological activities showed that the HS-binding N-terminal BMP2/4 and C-terminal BMP5/6/7 peptides stimulated chondrogenesis in vitro, potentially by freeing endogenous BMPs. Thus, HS interactions appear largely ascribable to domains at opposite ends of BMP2/4 versus BMP5/6/7, reiterating the evolutionary distance of these BMP subgroups and possible functional diversification.

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“…This interaction orients K114 in an optimal conformation to form hydrogen bonds and salt bridges with sugar residues at position +1 and +2 (Fig. 4) (Richard et al, 2009). …”

Section: Biochemical and Physiological Implications Of 3-o-sulfationmentioning

confidence: 99%

Heparan sulfate 3-O-sulfation: A rare modification in search of a function

Thacker¹,

Xu²,

Lawrence³

et al. 2014

Matrix Biology

199

244

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Many protein ligands bind to heparan sulfate, which results in their presentation, protection, oligomerization or conformational activation. Binding depends on the pattern of sulfation and arrangement of uronic acid epimers along the chains. Sulfation at the C3 position of glucosamine is a relatively rare, yet biologically significant modification, initially described as a key determinant for binding and activation of antithrombin and later for infection by Type I Herpes Simplex virus. In mammals, a family of seven heparan sulfate 3-O-sulfotransferases installs sulfate groups at this position and constitutes the largest group of sulfotransferases involved in heparan sulfate formation. However, to date very few proteins or biological systems have been described that are influenced by 3-O-sulfation. This review describes our current understanding of the prevalence and structure of 3-O-sulfation sites, expression and substrate specificity of the 3-O-sulfotransferase family and the emerging roles of 3-O-sulfation in biology.

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The Signature 3-O-Sulfo Group of the Anticoagulant Heparin Sequence Is Critical for Heparin Binding to Antithrombin but Is Not Required for Allosteric Activation

Cited by 42 publications

References 46 publications

Investigating changes in the gas-phase conformation of Antithrombin III upon binding of Arixtra using traveling wave ion mobility spectrometry (TWIMS)

Investigating changes in the gas-phase conformation of Antithrombin III upon binding of Arixtra using traveling wave ion mobility spectrometry (TWIMS)

Domains with highest heparan sulfate–binding affinity reside at opposite ends in BMP2/4 versus BMP5/6/7: Implications for function

Heparan sulfate 3-O-sulfation: A rare modification in search of a function

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