¹H NMR assignment and secondary structure of recombinant RGD‐hirudin

Abstract

The conformation of a new recombinant RGD-hirudin, which has the activities of anti-thrombin and anti-platelet aggregation, was investigated by multi-dimensional NMR spectroscopy. The 1H NMR spectra of this protein are assigned in a sequential manner by using a combination of 2D NMR techniques to demonstrate through-bond and through-space (<5 A) connectivities. The secondary structure of recombinant RGD-hirudin was deduced from chemical shift indices, sequential NOEs and 3J(HNalpha) coupling constants. The results show that the recombinant RGD-hirudin has two anti-parallel beta-sheets and no alpha-helix, and also that the Arg-Gly-Asp (RGD) binding motif of this protein is located at the end of a long arm, which consists of two anti-parallel beta-strands (residues 26-31 and 36-41). As the strands are connected by a beta-turn, the recombinant RGD-hirudin acquires high flexibility and inhibits platelet aggregation more effectively.

Hirudin or hirudin‐like factor ‐ that is the question: insights from the analyses of natural and synthetic HLF variants

“…Incorporation of an RGD motif by replacement of Ser 32 ‐Asp 33 ‐Gly 34 with the tripeptide Arg‐Gly‐Asp between cysteine residues 5 and 6 of hirudin variant HV1 did not alter its antithrombin activity . The RGD motif acts as a recognition site for integrin binding and facilitates the inhibition of platelet aggregation by the leech factors ornatin , decorsin , and RGD–hirudin .…”

Overproduction of 15N-Labeled r-RGD-Hirudin in Pichia pastoris for NMR Studies

“…The dispersion of the signals in the 1 H dimension covers the whole spectral area. As the proton resonances in TOCSY and NOESY spectra were assigned for r-RGD-hirudin [6], the assignment of 15 N-1 H signals can be performed more easily. A 3D 1 H-15 N edited HSQC-TOCSY spectrum [27] was acquired to correlate the 15 N with C H and side-chain protons.…”

“…Continuous structure studies have focused on the structure-function relationship of r-RGD-hirudin. The solution structure of r-RGD-hirudin was determined by 1 H NMR spectroscopy; on the basis of this structure, Song et al suggested a mechanism for r-RGDhirudin/thrombin binding and suggested that the r-RGD hi- [6,7]. The precise mechanisms and sites of interaction between r-RGD-hirudin and its receptors remain unknown.…”

“…Mass spectroscopy confirmed the uniform incorporation of 15 N. The efficient 15 N-labeling of r-RGD-hirudin enables preliminary NMR investigation, the results of which were a well-resolved 1 H-15 N HSQC spectrum. Based on the 3D 1 H-15 N edited HSQC-TOCSY and the results of 1 H NMR experiments [6,7], most of the signals in HSQC spectrum were assigned, clearing the path for detailed study of the interactions between r-RGD-hirudin and its receptors.…”

Proton Bridging in Catalysis by and Inhibition of Serine Proteases of the Blood Cascade System

“…Previous structural information from X-ray studies had revealed at least 13 sites near the cognate substrate-recognition site where hirudin might form short proton bridges. To aid locating the sites of possible short proton bridges between thrombin and hirudin, we employed several analogs of hirudin: hirunorm IV and hirunorm V [ 48 , 53 , 54 ], r-RGD-hirudin (recombinant 32SGD34 type 2 hirudin) [ 51 ] and an Nα(Me)Arg-peptide [ 52 ]. The hirunorms have three amino acids of the N-terminal sequence of hirudin followed by a linker and 10 amino acids of the C-terminus of hirudin, but are about hundred times less effective inhibitors of α-thrombin than the parent compound.…”