Computationally accessible method for estimating free energy changes resulting from site-specific mutations of biomolecules: Systematic model building and structural/hydropathic analysis of deoxy and oxy hemoglobins

Doctor

Carta

et al. 2006

Hendra virus (HeV) is a recently identified paramyxovirus that is fatal in humans and could be used as an agent of bioterrorism. The HeV receptor-binding protein (G) is required in order for the fusion protein (F) to mediate fusion, and analysis of the triggering/activation of HeV F by G should lead to strategies for interfering with this key step in viral entry. HeV F, once triggered by the receptor-bound G, by analogy with other paramyxovirus F proteins, undergoes multistep conformational changes leading to a six-helix bundle (6HB) structure that accomplishes fusion of the viral and cellular membranes. The ectodomain of paramyxovirus F proteins contains two conserved heptad repeat regions (HRN and HRC) near the fusion peptide and the transmembrane domains, respectively. Peptides derived from the HRN and HRC regions of F are proposed to inhibit fusion by preventing F, after the initial triggering step, from forming the 6HB structure that is required for fusion. HeV peptides have previously been found to be effective at inhibiting HeV fusion. However, we found that a human parainfluenza virus 3 F-peptide is more effective at inhibiting HeV fusion than the comparable HeV-derived peptide.

Section: Cells and Virusesmentioning

confidence: 65%

Inhibition of Hendra Virus Fusion

Doctor

Carta

et al. 2006

“…The relative energies of the bound ligands in the molecular models were evaluated with the HINT program (13,15,31). This program uses a non-Newtonian force field derived from experimental measurements of solvent partitioning to calculate free energy scores that can be converted to binding free energies (1,5,8,9). In this work, HINT 3.10S, which includes a number of local modifications (14,30), was used to calculate binding scores.…”

Section: Methodsmentioning

confidence: 99%

A Second Receptor Binding Site on Human Parainfluenza Virus Type 3 Hemagglutinin-Neuraminidase Contributes to Activation of the FusionMechanism

Fornabaio

Kellogg

et al. 2007

Self Cite

184

The hemagglutinin-neuraminidase (HN) protein of paramyxoviruses carries out three discrete activities that each affect the ability of HN to promote viral fusion and entry: receptor binding, receptor cleaving (neuraminidase), and triggering of the fusion protein. The interrelationship between the receptor binding and fusiontriggering functions of HN has not been clear. For human parainfluenza type 3 (HPIV3), one bifunctional site on HN can carry out both receptor binding and neuraminidase activities, and this site's receptor binding can be inhibited by the small receptor analog zanamivir. We now report experimental evidence, complemented by computational data, for a second receptor binding site near the HPIV3 HN dimer interface. This second binding site can mediate receptor binding even in the presence of zanamivir, and it differs from the second receptor binding site of the paramyxovirus Newcastle disease virus in its function and its relationship to the primary binding site. This second binding site of HPIV3 HN is involved in triggering F. We suggest that the two receptor binding sites on HPIV3 HN each contribute in distinct ways to virus-cell interaction; one is the multifunctional site that contains both binding and neuraminidase activities, and the other contains binding activity and also is involved in fusion promotion.Paramyxoviruses, including the human parainfluenza viruses, possess an envelope protein hemagglutinin-neuraminidase (HN) that has receptor cleaving as well as receptor binding activity. HN is also essential for activating the fusion protein (F) to mediate the merger of the viral envelope with the host cell membrane. For the parainfluenza viruses as well as other HN-containing paramyxoviruses, this one molecule carries out three different but critical activities at specific points in the process of viral entry. A clear mechanistic understanding of how these activities are regulated is key for the design of strategies to block viral entry (21). The specific role of HN in triggering F is a key question and has eluded simple explanations (3); we propose that a second binding site on HN exists and serves an important function in the triggering mechanism.Crystallographic studies of the human parainfluenza virus type 3 (HPIV3) HN (17), the Newcastle disease virus (NDV) HN (6,33), and the simian virus 5 HN (32) indicated that a single active/catalytic site on the globular head of the HN molecule can indeed provide both receptor binding and neuraminidase activities. For NDV, experimental evidence for the importance of this site ("site I") to receptor binding includes the finding that mutations in site I alter receptor binding (2, 18). For HPIV3, mutations at the putative receptor binding site also affect receptor binding avidity (22,23,25). However, the effects of the receptor analog zanamivir on activities of the two HN molecules were different: for HPIV3 HN, both receptor binding and neuraminidase activity were blocked by zanamivir, while for NDV HN, receptor binding was partially resistant to the e...

“…The wells were then washed three times with 300 or 150 l cold CO 2 -independent medium (catalog number 18045-088; Gibco, Gaithersburg, MD). The bound RBCs were lysed with 200 l RBC lysis solution (0.145 M NH 4 Cl, 17 mM Tris HCl), and the absorbance was read at 540 nm on the Bio-Tek ELISA reader.…”

Section: Cellsmentioning

confidence: 99%

“…The plates were placed at 4°C for 30 min to allow RBC binding. The cell monolayers were then washed at 4°C with cold CO 2 -independent medium to remove unbound RBCs, the bound RBCs were lysed (0.145 M NH 4 Cl, 17 mM Tris HCl), and the absorbance was read at 540 nm on the Bio-Tek ELISA reader. Results are presented as the percent retention of RBCs relative to control (undepleted RBCs) versus the degree of depletion expressed as milliunits of bacterial neuraminidase.…”

Section: Cellsmentioning

confidence: 99%

“…The relative energies of the bound ligands in the molecular models were evaluated with the HINT (Hydropathic INTeractions; Tripos, Inc., St. Louis, MO) program (14,16,27). This program uses a unique non-Newtonian force field derived from experimental measurements of solvent partitioning to calculate free-energy scores that can be readily converted to binding free energies (4,7,10,11). In this work, HINT 3.10S, which includes a number of local modifications (15,26), was used to calculate binding scores.…”

Section: Cellsmentioning

confidence: 99%

See 1 more Smart Citation

Paramyxovirus Receptor-Binding Molecules: Engagement of One Site on the Hemagglutinin-Neuraminidase Protein Modulates Activity at the Second Site

Fornabaio

Greengard

et al. 2006

Self Cite

The hemagglutinin-neuraminidase (HN) protein of paramyxoviruses carries out three different activities: receptor binding, receptor cleaving (neuraminidase), and triggering of the fusion protein. These three discrete properties each affect the ability of HN to promote viral fusion and entry. For human parainfluenza type 3, one bifunctional site on HN can carry out both binding and neuraminidase, and the receptor mimic, zanamivir, impairs viral entry by blocking receptor binding. We report here that for Newcastle disease virus, the HN receptor avidity is increased by zanamivir, due to activation of a second site that has higher receptor avidity. Only certain receptor mimics effectively activate the second site (site II) via occupation of site I; yet without activation of this second site, binding is mediated entirely by site I. Computational modeling designed to complement the experimental approaches suggests that the potential for small molecule receptor mimics to activate site II, upon binding to site I, directly correlates with their predicted strengths of interaction with site I. Taken together, the experimental and computational data show that the molecules with the strongest interactions with site I-zanamivir and BCX 2798-lead to the activation of site II. The finding that site II, once activated, shows higher avidity for receptor than site I, suggests paradigms for further elucidating the regulation of HNs multiple functions in the viral life cycle.Paramyxoviruses, including human parainfluenza virus type 3 (HPIV3) and the avian paramyxovirus Newcastle disease virus (NDV), possess an envelope protein hemagglutinin-neuraminidase (HN) that has receptor-cleaving as well as receptorbinding activity. HN is also essential for activating the fusion protein (F) to mediate the merger of the viral envelope with the host cell membrane. For both HPIV3 and NDV, this one molecule carries out three different but critical activities at specific points in the process of viral entry, and understanding the regulation of these activities is key for the design of strategies that block viral entry (19).We have previously used a small molecule receptor mimic, zanamivir (4-guanidino-neu5Ac2en [DANA]), to probe paramyxovirus active sites and found distinctions between HPIV3 HN and NDV HN that are manifested by differences in the sensitivity of individual HN functions to zanamivir. Zanamivir can reversibly occupy the neuraminidase active site/receptor-binding site of HPIV3 HN (17). The compound was originally developed as an influenza neuraminidase inhibitor and has a sialidase K i (M) of approximately 1 ϫ 10 Ϫ9 for the influenza A neuraminidase (13). The K i for the parainfluenza virus neuraminidases is lower, approximately 8 ϫ 10 Ϫ4 for HPIV2, the virus for which values have been obtained (13).For HPIV3, zanamivir inhibited both the receptor-binding function and the neuraminidase function of HN (12) but had its primary anti-infective effect by blocking receptor binding and thus impairing entry. A mutation at the active site (T193I...