Nuclear Magnetic Resonance Structural and Ligand Binding Studies of BLBC, a Two-Domain Fragment of Barley Lectin

Abstract

Plant lectins are useful targets for biophysical studies of protein-carbohydrate recognition, a process of general interest because of its many roles in human physiology. Here, nuclear magnetic resonance (NMR) based structural and carbohydrate binding data on a two-domain fragment of the normally four-domain barley lectin protein are presented. The structural data, while preliminary, clearly shows that the recombinantly produced simplified model system, called BLBC, retains a nativelike fold. However, unlike the full-length parent protein, which is dimeric, BLBC is shown by pulsed-field gradient NMR diffusion studies to be largely monomeric. Still, the fragment retains nativelike carbohydrate binding properties. These properties are examined in some detail using heteronuclear single quantum coherence (HSQC) NMR spectroscopy on a uniformly 15N-labeled sample. Ligand-induced chemical shift changes in the 1H-15N HSQC spectrum are monitored as 15N-labeled BLBC is titrated with increasing concentrations of the unlabeled carbohydrate, N,N',N"-triacetylchitotriose. Well-resolved resonances from the individual domains show that BLBC binds ligand at two distinct and independent ligand binding sites, one in each domain. Binding constants of (1.1 +/- 0.2) x 10(3) M-1 and (0.6 +/- 0.2) x 10(3) M-1 are determined for the B and C domain sites, respectively. These results are discussed in relation to ligand binding studies that have previously been carried out on a highly homologous protein, wheat germ agglutinin.

Order Matrix Analysis of Residual Dipolar Couplings Using Singular Value Decomposition

“…The 89-residue BLBC fragment shares approximately 95% sequence homology with wheat germ agglutinin, WGA, which has been the subject of numerous crystal structure studies (32). Previous NOE-based studies of BLBC were not sufficient to determine a high-resolution structure, but they did show the domains to have folds very similar to those seen in the crystal structure of WGA, and they did indicate that the center part of each domain was well structured (33). More significantly for our purposes, the relative orientation of the B and C domains of BLBC could not be determined because of the lack of long-range restraints.…”

Diffusion NMR spectroscopy

“…This property is widely exploited in in vitro MRS studies of biomolecules in solution to assess various biophysical and biochemical parameters, including the hydrodynamics and the folding/unfolding of proteins, 10 proton exchange with biomacromolecules, 11 the oligomerization state of biomacromolecules 12 and protein-ligand interactions. 13 Equation (8) shows that diffusion strongly depends on temperature which should therefore ideally be kept constant throughout the diffusion measurement, unless temperature per se is the parameter of interest. The diffusion coefficient D exhibits a non-linear dependence on the temperature T because the viscosity Z in eqn (9) is strongly affected by T. This results in an exponential dependence of D on T:…”

“…2 is inevitably affected by T 2 relaxation. Equation (13) implies that the dephasing efficiency of a gradient pulse depends on the type of nucleus (the higher the g, the stronger the dephasing effect will be), the strength and duration of the gradient pulse, and the displacement of the spin along the gradient direction. Although the example of eqns (12) and (13) In order to appreciate the effects of diffusion on the signal intensity from an ensemble of nuclear spins, it is necessary to consider the probability of a spin starting at r 0 to move to r 1 in the time D. This probability is given by P(r 0 jr 1 ,t) as before [see eqn (5)].…”

“…the probability of the molecular translational displacement; equivalent to eqn (7)] is determined directly by Fourier transformation of the attenuation of the MR signal (at fixed PFG timing) with respect to q, the spatial wave number. The q-vector that is defined as (g g)/2p (in units of cm À1 ), determines the value of the diffusion-induced phase shift [see eqn (13)]. This type of analysis enables the estimation of the existence of restricted diffusion and the estimation of the compartment size.…”

Structural Basis for New Pattern of Conserved Amino Acid Residues Related to Chitin-binding in the Antifungal Peptide from the Coconut Rhinoceros Beetle Oryctes rhinoceros

“…The plant chitin-binding domain consists mostly of 30 -43 residues, including eight cysteines, three aromatic residues, and glycines, and is frequently referred to as a hevein domain (4). This domain is indispensable to antimicrobial activity and shows significant conservation in the primary sequence (Ͼ40%) and in the tertiary structure (5)(6)(7)(8). Although chitin is an important structural component of invertebrates, fungi, and bacteria, chitin-binding proteins of invertebrates have only recently been characterized (9 -13).…”