Multiple Receptors Involved in Human Rhinovirus Attachment to Live Cells Christian Rankl, Ferry Kienberger, Linda Wildling, Jürgen Wruss, Hermann J. Gruber, Dieter Blaas, and Peter Hinterdorfer

Background 12/100 HRV serotypes (minor group) bind LDLR/VLDLR/ LRP Receptors involved in endocytosis / signal transduction Initial binding EDTA labile Subsequent step (after several minutes) non-dissociable

LDLR/ VLDLR MBP-VLDR1-8-(his)6 40 aa ligand modules, stabilized by a Ca ion and 6 Cys

AFM is a very high-resolution type of scanning probe microscope, with demonstrated resolution of fractions of 1nm, > 1000 times optical diffraction limit. AFM consists of a cantilever (nm size) with a sharp tip (probe) at its end that is used to scan the specimen surface. When the tip is brought into proximity of a sample surface, forces between the tip and the sample lead to a deflection of the cantilever according to Hooke’s law F = k x d A major application of AFM is force spectroscopy: measurement of force-distance curves. The AFM tip is extended towards and retracted from the surface as the static deflection of the cantilever is monitored as a function of pizoelectric displacement. Can measure pN forces (single molecule) Scanner has 3D positioning (<nm res.) Atomic force microscopy (AFM)

AFM For single-molecule interactions: 1.Binding to surface should be covalent (1 -9 N) - much higher than receptor-ligand interaction 2.Surface density of receptors should be low enough 3.Molecules should retain mobility 4.Non-specific adsorption inhibited 5.Molecules should be oriented

Figure 1 NHS benzaldehyde 1 2

Unbinding force

Figure 2 Single molecule: 82 +/- 14pN At 300nm/s

Figure 3 PDF: probability density functions

Figure 4 KD= 24nM

Figure 5 Single molecule: 22 pN At 1500nm/s

Figure 6

Conclusions Chemistry allows free movement of receptor and ligand Data indicates multiple receptors binding to each virus Increasing contact time increased interaction KD = 24nM Cell expt. Showed KD 10X lower