PDBsum entry 2let

Proteinase inhibitor(trypsin)

PDB id

2let

Contents

			Protein chain

					28 a.a.

References listed in PDB file

Key reference

Title

An 1h nmr determination of the three-Dimensional structures of mirror-Image forms of a leu-5 variant of the trypsin inhibitor from ecballium elaterium (eeti-Ii).

Authors

K.J.Nielsen, D.Alewood, J.Andrews, S.B.Kent, D.J.Craik.

Ref.

Protein Sci, 1994, 3, 291-302. [DOI no: 10.1002/pro.5560030213]

PubMed id

8003965

Note In the PDB file this reference is annotated as "TO BE PUBLISHED". The citation details given above were identified by an automated search of PubMed on title and author names, giving a percentage match of 87%.

Abstract

The 3-dimensional structures of mirror-image forms of a Leu-5 variant of the trypsin inhibitor Ecballium elaterium (EETI-II) have been determined by 1H NMR spectroscopy and simulated annealing calculations incorporating NOE-derived distance constraints. Spectra were assigned using 2-dimensional NMR methods at 400 MHz, and internuclear distances were determined from NOESY experiments. Three-bond spin-spin couplings between C alpha H and amide protons, amide exchange rates, and the temperature dependence of amide chemical shifts were also measured. The structure consists largely of loops and turns, with a short region of beta-sheet. The Leu-5 substitution produces a substantial reduction in affinity for trypsin relative to native EETI-II, which contains an Ile at this position. The global structure of the Leu-5 analogue studied here is similar to that reported for native EETI-II (Heitz A, Chiche L, Le-Nguyen D, Castro B, 1989, Biochemistry 28:2392-2398) and to X-ray and NMR structures of the related proteinase inhibitor CMTI-I (Bode W et al., 1989, FEBS Lett 242:285-292; Holak TA et al., 1989a, J Mol Biol 210:649-654; Holak TA, Gondol D, Otlewski J, Wilusz T, 1989b, J Mol Biol 210:635-648; Holak TA, Habazettl J, Oschkinat H, Otlewski J, 1991, J Am Chem Soc 113:3196-3198). The region near the scissile bond is the most disordered part of the structure, based on geometric superimposition of 40 calculated structures. This disorder most likely reflects additional motion being present in this region relative to the rest of the protein. This motional disorder is increased in the Leu-5 analogue relative to the native form and may be responsible for its reduced trypsin binding. A second form of the protein synthesized with all (D) amino acids was also studied by NMR and found to have a spectrum identical with that of the (L) form. This is consistent with the (D) form being a mirror image of the (L) form and not distinguishable by NMR in an achiral solvent (i.e., H2O). The (D) form has no activity against trypsin, as would be expected for a mirror-image form.



	Figure 1. Fig. 1. rimarystructure of sowingthediulfideconnectiv- ities.		Figure 5. Fig. 5. Region of 120-ms TOCSYspectrum of Leu5-EETI-IIin D20 showingthe slow exchane NHprotonconnectivities to heirsidechainprotons.

PROCHECK

	Headers