Structural studies of neuropilin/antibody complexes provide insights into semaphorin and VEGF binding.

Neuropilins (Nrps) are co-receptors for class 3 semaphorins and vascular endothelial growth factors and important for the development of the nervous system and the vasculature. The extracellular portion of Nrp is composed of two domains that are essential for semaphorin binding (a1a2), two domains necessary for VEGF binding (b1b2), and one domain critical for receptor dimerization (c). We report several crystal structures of Nrp1 and Nrp2 fragments alone and in complex with antibodies that selectively block either semaphorin or vascular endothelial growth factor (VEGF) binding. In these structures, Nrps adopt an unexpected domain arrangement in which the a2, b1, and b2 domains form a tightly packed core that is only loosely connected to the a1 domain. The locations of the antibody epitopes together with in vitro experiments indicate that VEGF and semaphorin do not directly compete for Nrp binding. Based upon our structural and functional data, we propose possible models for ligand binding to neuropilins.

The high-resolution structures of the neutral and the low pH crystals of aminopeptidase from Aeromonas proteolytica.

The aminopeptidase from Aeromonas proteolytica (AAP) contains two zinc ions in the active site and catalyzes the degradation of peptides. Herein we report the crystal structures of AAP at 0.95-A resolution at neutral pH and at 1.24-A resolution at low pH. The combination of these structures allowed the precise modeling of atomic positions, the identification of the metal bridging oxygen species, and insight into the physical properties of the metal ions. On the basis of these structures, a new putative catalytic mechanism is proposed for AAP that is likely relevant to all binuclear metalloproteases.

Suppressor of fused regulates Gli activity through a dual binding mechanism.

The Hedgehog pathway drives proliferation and differentiation by activating the Gli/Ci family of zinc finger transcription factors. Gli/Ci proteins form Hedgehog signaling complexes with other signaling components, including the kinesin-like protein Costal-2, the serine-threonine kinase Fused, and Suppressor of Fused [Su(fu)]. In these complexes Gli/Ci proteins are regulated by cytoplasmic sequestration, phosphorylation, and proteolysis. Here we characterize structural and functional determinants of Su(fu) required for Gli regulation and show that Su(fu) contains at least two distinct domains: a highly conserved carboxy-terminal region required for binding to the amino-terminal ends of the Gli proteins and a unique amino-terminal domain that binds the carboxy-terminal tail of Gli1. While each domain is capable of binding to different Gli1 regions independently, interactions between Su(fu) and Gli1 at both sites are required for cytoplasmic tethering and repression of Gli1. Furthermore, we have solved the crystal structure of the amino-terminal domain of human Su(fu)(27-268) at 2.65 A resolution. This domain forms a concave pocket with a prominent acidic patch. Mutation at Asp(159) in the acidic patch disrupts Gli1 tethering and repression while not strongly disrupting binding, indicating that the amino-terminal domain of Su(fu) likely impacts Gli binding through a mechanism distinct from that for tethering and repression. These studies provide a structural basis for understanding the function of Su(fu).

The 1.20 A resolution crystal structure of the aminopeptidase from Aeromonas proteolytica complexed with tris: a tale of buffer inhibition.

The aminopeptidase from Aeromonas proteolytica (AAP) is a bridged bimetallic enzyme that removes the N-terminal amino acid from a peptide chain. To fully understand the metal roles in the reaction pathway of AAP we have solved the 1.20 A resolution crystal structure of native AAP (PDB ID = 1LOK). The high-quality electron density maps showed a single Tris molecule chelated to the active site Zn(2+), alternate side chain conformations for some side chains, a sodium ion that mediates a crystal contact, a surface thiocyanate ion, and several potential hydrogen atoms. In addition, the high precision of the atomic positions has led to insight into the protonation states of some of the active site amino acid side chains.

Attempted Syntheses of Transition Metal and Lanthanide (Dialkylamino)squarates. The Hydrolysis Problem.

Reaction of 1-amino-2-methoxycyclobutenedione with M(NO(3))(2).xH(2)O (M = Mn, Co, Ni, Cu, Zn) in aqueous solution results in the formation of the squarates M(C(4)O(4)).4H(2)O and/or M(C(4)O(4)).2H(2)O owing to the hydrolysis of both the methoxy and amino groups on the ligand. Similarly, reaction of this ligand with M(NO(3))(3).6H(2)O (M = La, Eu, Gd, Tb) results in the formation of the respective lanthanide squarates. Buffering the reactant solutions at different pH's did not change the results and neither did using the alternative solvents methanol, ethanol, propan-1-ol, and acetonitrile. In previously reported reactions between 1-amino-2-methoxycyclobutenedione and 1-(dimethylamino)-2-methoxycyclobutenedione separately with Pb(NO(3))(2) under similar reaction conditions, the amino groups remained unhydrolyzed. The substituent amino groups also remain unhydrolyzed when these ligands are left to stand in aqueous solution in the absence of transition metal ions; only the methoxy group is hydrolyzed. 1-(Dimethylamino)-2-hydroxycyclobutenedione (1), formed from such a hydrolysis of 1-(dimethylamino)-2-methoxycyclobutenedione in aqueous solution, crystallizes in space group P2(1)/c, with a = 5.093(1) Å, b = 8.331(1) Å, c = 15.087(3) Å, beta = 95.12(1) degrees, and Z = 4. However, attempts at complexation of the ligand with Mn(II) ions resulted in the formation of a mixture of the Mn(II) squarate Mn(C(4)O(4)).4H(2)O and the salt [Mn(H(2)O)(6)][HC(4)O(3)NH(2)](2).2H(2)O (2), in which the amino group remains intact. Compound 2 crystallizes in the tetragonal space group P4(2)/m with a = 7.251(3) Å, c = 15.979(8) Å, and Z = 2. If tetraethylammonium aminosquarate is used instead of 1-amino-2-methoxycyclobutenedione, both [Co(C(4)O(4))(H(2)O)(4)] and [Co(C(4)O(4))(H(2)O)(2)] are formed on reaction with Co(NO(3))(2).6H(2)O. Hydrolysis of the amino group also occurs when the higher homologue 1-(dimethylamino)-2-methoxycyclobutenedione is reacted with Co(NO(3)).6H(2)O, but only [Co(C(4)O(4))(H(2)O)(2)] is formed. [Co(C(4)O(4))(H(2)O)(2)] crystallizes in the space group Pn&thremacr;n with a = 16.256(1) Å and Z = 24 and is isomorphous with [Co(C(4)O(4))(H(2)O)(2)].0.33H(2)O, which has been reported previously. Evidence suggests that the hydrolysis of the dialkylamino substituents in the 1-(dialkylamino)-2-methoxycyclobutenediones is apparently mediated by the transition and lanthanide metals. The use of the hydrolysis of the amino group in 1-dialkylamino derivatives of squaric acid as a convenient synthetic tool is discussed.